Plants and methods to improve agronomic characteristics under abioticstress conditions

Abstract

Isolated polynucleotides and polypeptides, and recombinant DNA constructs useful for conferring improved drought tolerance; compositions (such as plants or seeds) comprising these recombinant DNA constructs; and methods utilizing these recombinant DNA constructs are disclosed. The recombinant DNA constructs comprise a polynucleotide operably linked to a promoter that is functional in a plant, wherein said polynucleotides encode drought tolerance polypeptides.

Description

FIELD

The field relates to plant breeding and genetics and, in particular, relates to recombinant DNA constructs useful in plants for improving tolerance to abiotic stress, such as drought stress.

BACKGROUND

Stresses to plants may be caused by both biotic and abiotic agents. For example, biotic causes of stress include infection with pathogen, insect feeding, and parasitism by another plant such as mistletoe. Abiotic stresses include, for example, excessive or insufficient available water, temperature extremes, and synthetic chemicals such as herbicides.

Abiotic stress is the primary cause of crop loss worldwide, causing average yield losses of more than 50% for major crops (Boyer, J. S. (1982) Science 218:443-448; Bray, E. A. et al. (2000) In Biochemistry and Molecular Biology of Plants, edited by Buchannan, B. B. et al., Amer. Soc. Plant Biol., pp. 1158-1249). Plants are sessile and have to adjust to the prevailing environmental conditions of their surroundings. This has led to their development of a great plasticity in gene regulation, morphogenesis, and metabolism. Adaption and defense strategies involve the activation of genes encoding proteins important in the acclimation or defense towards the different stresses.

Drought (insufficient available water) is one of the major abiotic stresses that limit crop productivity worldwide, and exposure of plants to a water-limiting environment during various developmental stages appears to activate various physiological and developmental changes. Although many reviews on molecular mechanisms of abiotic stress responses and genetic regulatory networks of drought stress tolerance have been published (Valliyodan, B., and Nguyen, H. T. (2006) Curr. Opin. Plant Biol. 9:189-195; Wang, W., et al. (2003) Planta 218:1-14; Vinocur, B., and Altman, A. (2005) Curr. Opin. Biotechnol. 16: 123-132; Chaves, M. M., and Oliveira, M. M. (2004) J. Exp. Bot. 55: 2365-2384; Shinozaki, K., et al. (2003) Curr. Opin. Plant Biol. 6:410-417; Yamaguchi-Shinozaki, K., and Shinozaki, K. (2005) Trends Plant Sci. 10:88-94), it remains a major challenge in biology to understand the basic biochemical and molecular mechanisms of drought stress perception, signal transduction and tolerance. Genetic research has shown that drought tolerance is a quantitative trait, controlled by many genes. Molecular marker-assisted breeding has led to improved drought tolerance in crops. However, marker accuracy and breeding efficiency remain problematic (Ashraf M. (2010) Biotechnol. Adv. 28:169-183). The transgenic approaches to engineering drought tolerance in crops have made great progress (Vinocur B. and Altman A. (2005) Curr. Opin. Biotechnol. 16:123-132; Lawlor D W. (2013) J. Exp. Bot. 64:83-108).

Earlier work on molecular aspects of abiotic stress responses was accomplished by differential and/or subtractive analysis (Bray, E. A. (1993) Plant Physiol. 103:1035-1040; Shinozaki, K., and Yamaguchi-Shinozaki, K. (1997) Plant Physiol. 115:327-334; Zhu, J.-K. et al. (1997) Crit. Rev. Plant Sci. 16:253-277; Thomashow, M. F. (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50:571-599); and other methods which include selection of candidate genes and analysis of expression of such a gene or its active product under stresses, or by functional complementation in a stressor system that is well defined (Xiong, L. and Zhu, J.-K. (2001) Physiologia Plantarum 112:152-166). Additionally, forward and reverse genetic studies involving the identification and isolation of mutations in regulatory genes have been used to provide evidence for observed changes in gene expression under stress (Xiong, L. and Zhu, J.-K. (2001) Physiologia Plantarum 112:152-166).

Activation tagging can be utilized to identify genes with the ability to affect a trait, and this approach has been used in Arabidopsis thaliana (the model plant species) (Weigel, D., et al. (2000) Plant Physiol. 122:1003-1013). Insertions of transcriptional enhancer elements can dominantly activate and/or elevate the expression of nearby endogenous genes, so it can be used to select genes involved in agronomically important phenotypes, including abiotic stress tolerance such as improved drought tolerance.

SUMMARY

The following embodiments are among those encompassed by the disclosure:

In one embodiment, the present disclosure includes an isolated polynucleotide, comprising: (a) a polynucleotide with nucleotide sequence of at least 85% sequence identity to SEQ ID NO: 3, 6, 9, 12, 15 or 18; (b) a polynucleotide with nucleotide sequence of at least 85% sequence identity to SEQ ID NO: 4, 7, 10, 13, 16 or 19; (c) a polynucleotide encoding a polypeptide with amino acid sequence of at least 90% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20; or (d) the full complement of the nucleotide sequence of (a), (b) or (c), wherein over-expression of the polynucleotide in a plant enhances drought tolerance; the isolated polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18 or 19; and the said polypeptide comprises the amino acid sequence of SEQ ID NO: 5, 8, 11, 14, 17 or 20.

In another embodiment, the present disclosure includes a recombinant DNA construct comprising the isolated polynucleotide operably linked to at least one heterologous regulatory sequence, wherein the polynucleotide comprises (a) a polynucleotide with nucleotide sequence of at least 85% sequence identity to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18 or 19; (b) a polynucleotide encoding a polypeptide with amino acid sequence of at least 90% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20; or (c) the full complement of the nucleotide sequence of (a) or (b).

In another embodiment, the present disclosure includes a transgenic plant or seed comprising a recombinant DNA construct, wherein the recombinant DNA construct comprises the polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide comprises (a) a polynucleotide with nucleotide sequence of at least 85% sequence identity to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18 or 19; (b) a polynucleotide encoding a polypeptide with amino acid sequence of at least 90% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20; or (c) the full complement of the nucleotide sequence of (a) or (b).

In another embodiment, the present disclosure includes a transgenic plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein the polynucleotide comprises (a) a polynucleotide with nucleotide sequence of at least 85% sequence identity to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18 or 19; (b) a polynucleotide encoding a polypeptide with amino acid sequence of at least 90% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20; or (c) the full complement of the nucleotide sequence of (a) or (b); the said plant exhibits improved drought tolerance when compared to a control plant.

In another embodiment, the present disclosure includes any of the plants of the disclosure, wherein the plant is selected from the group consisting of rice, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, barley, millet, sugar cane and switchgrass.

In another embodiment, methods are provided for increasing drought tolerance in a plant, comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, when compared to SEQ ID NO: 5, 8, 11, 14, 17 or 20; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (c) obtaining a progeny plant derived from the transgenic plant of step (b), wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits increased drought tolerance when compared to a control plant not comprising the recombinant DNA construct.

In another embodiment, methods are provided for evaluating drought tolerance in a plant, comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, when compared to SEQ ID NO: 5, 8, 11, 14, 17 or 20; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; (c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (d) evaluating the progeny plant for drought tolerance compared to a control plant not comprising the recombinant DNA construct.

In another embodiment, the present disclosure concerns a recombinant DNA construct comprising any of the isolated polynucleotides of the present disclosure operably linked to at least one regulatory sequence, and a cell, a plant, or a seed comprising the recombinant DNA construct. The cell may be eukaryotic, e.g., a yeast, insect or plant cell; or prokaryotic, e.g., a bacterial cell.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The disclosure can be more fully understood from the following detailed description and the accompanying drawings and sequence listing which form a part of this application.

FIG. 1 shows changes of soil volumetric moisture content at different developmental stages in the first field experiment for drought screening OsGSTU41 transgenic rice. The OsGSTU41 transgenic rice started heading at 26 days after stopping watering and matured at 63 days after stopping watering.

FIG. 2 shows changes of soil volumetric moisture content at different developmental stages in the second field experiment for drought screening OsGSTU41 transgenic rice. The OsGSTU41 transgenic rice started heading at 43 days after stopping watering and matured at 73 days after stopping watering.

FIG. 3 shows the relative expression levels of OsGSTU41 gene in leaves of different transgenic rice lines by real-time PCR analyses. The base expression level in DP0043.15 is set at 1.00, the numbers on the top of the columns are fold-changes compared to DP0043.15 rice. ZH11 is wild type rice.

FIG. 4 shows the relative expression levels of OsPPCK4 gene in leaves of different transgenic rice lines by real-time PCR analyses. The base expression level in ZH11-TC is set at 1.00, the numbers on the top of the columns are fold-changes compared to ZH11-TC rice. ZH11-TC is tissue cultured ZH11 rice and DP0158 is empty vector transformed ZH11 rice plants.

FIG. 5 shows the relative expression levels of OsDN-DTP4 gene in leaves of different transgenic rice lines by real-time PCR analyses. The base expression level in ZH11-TC is set at 1.00, the numbers on the top of the columns are fold-changes compared to ZH11-TC rice. ZH11-TC is tissue cultured ZH11 rice and DP0158 is empty vector transformed ZH11 rice plants.

FIG. 6 shows the relative expression levels of OsLecRK4.1 gene in leaves of different transgenic rice lines by real-time PCR analyses. The base expression level in ZH11-TC is set at 1.00, the numbers on the top of the columns are fold-changes compared to ZH11-TC rice. ZH11-TC is tissue cultured ZH11 rice plants.

FIG. 7 shows the relative expression levels of OsLecRK4.2 gene in leaves of different transgenic rice lines by real-time PCR analyses. The base expression level in ZH11-TC is set at 1.00, the numbers on the top of the columns are fold-changes compared to ZH11-TC rice. ZH11-TC is tissue cultured ZH11 rice plants.

FIG. 8 shows changes of soil volumetric water content at different developmental stage for drought testing OsPPCK4 transgenic rice.

FIG. 9 shows changes of soil volumetric water content at different developmental stage for drought testing OsCAM2 transgenic rice in the first experiment. The OsCAM2 transgenic rice started heading at 31 days after stopping watering.

FIG. 10 shows changes of soil volumetric water content at different developmental stage for drought testing OsCAM2 transgenic rice in the second experiment. The OsCAM2 transgenic rice started heading at 24 days after stopping watering.

FIG. 11 shows changes of soil volumetric water content at different developmental stage for drought testing OsLecRK4.1 transgenic rice. The OsLecRK4.1 transgenic rice started heading at 22 days after stopping watering.

FIG. 12 shows changes of soil volumetric water content at different developmental stage for drought testing OsLecRK4.2 transgenic rice. The OsLecRK4.2 transgenic rice started heading at 25 days after stopping watering.

Table 1. SEQ ID NOs for nucleotide and amino acid sequences provided in the sequence listing

Table 2. Rice gene names, Gene IDs (from TIGR) and Construct IDs

Table 3. Primers for cloning rice drought tolerance genes

Table 4. PCR reaction mixture for cloning drought tolerance genes

Table 5. PCR cycle conditions

Table 6. Enhanced drought tolerance of OsGSTU41 transgenic rice plants under greenhouse conditions (1^st experiment)

Table 7. Enhanced drought tolerance of OsGSTU41 transgenic rice plants under greenhouse conditions at construct level (2^nd experiment)

Table 8. Enhanced drought tolerance of OsGSTU41 transgenic rice plants under greenhouse conditions at line level (2^nd experiment)

Table 9. Enhanced drought tolerance of OsPPCK4 transgenic rice plants under greenhouse conditions (1^st experiment)

Table 10. Enhanced drought tolerance of OsPPCK4 transgenic rice plants under greenhouse conditions at construct level (2^nd experiment)

Table 11. Enhanced drought tolerance of OsPPCK4 transgenic rice plants under greenhouse conditions at transgenic line level (2^nd experiment)

Table 12. Enhanced drought tolerance of OsCAM2 transgenic rice plants under greenhouse conditions (1^st experiment)

Table 13. Enhanced drought tolerance of OsCAM2 transgenic rice plants under greenhouse conditions at construct level (2^nd experiment)

Table 14. Enhanced drought tolerance of OsCAM2 transgenic rice plants under greenhouse conditions at line level (2^nd experiment)

Table 15. Enhanced drought tolerance of OsCAM2 transgenic rice plants under greenhouse conditions at line level (3^rd experiment)

Table 16. Enhanced drought tolerance of OsCAM2 transgenic rice plants under greenhouse conditions at line level (3^rd experiment)

Table 17. Drought tolerance assay of OsDN-DTP4 transgenic rice plants under greenhouse conditions (1^st experiment)

Table 18. Drought tolerance assay of OsDN-DTP4 transgenic rice plants under greenhouse conditions at construct level (2^nd experiment)

Table 19. Drought tolerance assay of OsDN-DTP4 transgenic rice plants under greenhouse conditions at line level (2^nd experiment)

Table 20. Drought tolerance assay of OsDN-DTP4 transgenic rice plants under greenhouse conditions at construct level (3^rd experiment)

Table 21. Drought tolerance assay of OsDN-DTP4 transgenic rice plants under greenhouse conditions at line level (3^rd experiment)

Table 22. Grain yield analysis of OsGSTU41 transgenic rice plants under field drought conditions (1^st experiment)

Table 23. Grain yield analysis of OsGSTU41 transgenic rice plants under field drought conditions (2^nd experiment)

Table 24. Grain yield analysis of OsPPCK4 transgenic rice plants under field drought conditions

Table 25. Grain yield assay of OsCAM2 transgenic rice plants under field drought conditions (1^st experiment)

Table 26. Grain yield assay of OsCAM2 transgenic rice plants under field drought conditions (2^nd experiment)

Table 27. Grain yield analysis of OsLecRK4.1 transgenic rice plants under field drought conditions

Table 28. Grain yield analysis of OsLecRK4.2 transgenic rice plants under field drought conditions

Table 29. Paraquat tolerance assay of OsGSTU41 transgenic rice plants at transgenic line level (1^st experiment)

Table 30. Paraquat tolerance assay of OsGSTU41 transgenic rice plants at transgenic line level (2^nd experiment)

Table 31. Paraquat tolerance assay of OsPPCK4 transgenic rice plants at transgenic line level (1^st experiment)

Table 32. Paraquat tolerance assay of OsPPCK4 transgenic rice plants at transgenic line level (2^nd experiment)

Table 33. Paraquat tolerance assay of OsPPCK4 transgenic rice plants at transgenic line level (3^rd experiment)

Table 34. Paraquat tolerance assay of OsCAM2 transgenic rice plants at transgenic line level (1^st experiment)

Table 35. Paraquat tolerance assay of OsCAM2 transgenic rice plants at transgenic line level (2^nd experiment)

Table 36. Paraquat tolerance assay of OsDN-DTP4-transgenic rice plant at transgenic line level (1^st experiment)

Table 37. Paraquat tolerance assay of OsDN-DTP4 transgenic rice plant at transgenic line level (2^nd experiment)

Table 38. Paraquat tolerance assay of OsLecRK4.1 transgenic rice plant at transgenic line level (1^st experiment)

Table 39. Paraquat tolerance assay of OsLecRK4.1 transgenic rice plant at transgenic line level (2^nd experiment)

Table 40. Paraquat tolerance assay of OsLecRK4.2 transgenic rice plant at transgenic line level (1^st experiment)

Table 41. Paraquat tolerance assay of OsLecRK4.2 transgenic rice plant at transgenic line level (2^nd experiment)

TABLE 1
SEQ ID NOs for nucleotide and amino acid sequences
provided in the sequence listing
		SEQ ID NO:	SEQ ID NO:
Source Species	Clone Designation	(Nucleotide)	(Amino Acid)
Artificial	DP0005 vector	1	n/a
Artificial	DsRed expression	2	n/a
Oryza sativa	OsGSTU41	3, 4	5
Oryza sativa	OsPPCK4	6, 7	8
Oryza sativa	OsCAM2	9, 10	11
Oryza sativa	OsDN-DTP4	12, 13	14
Oryza sativa	OsLecRK4.1	15, 16	17
Oryza sativa	OsLecRK4.2	18, 19	20
Artificial	Primers	21-42	n/a

The Sequence Listing contains the one-letter code for nucleotide sequences and the three-letter code for amino acid sequences as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R.§ 1.822.

SEQ ID NO: 1 is the nucleotide sequence of vector DP0005.

SEQ ID NO: 2 is the nucleotide sequence of DsRed expression cassette.

SEQ ID NO: 3 is the nucleotide sequence of cDNA of OsGSTU41 gene.

SEQ ID NO: 4 is the nucleotide sequence of CDS of OsGSTU41 gene.

SEQ ID NO: 5 is the amino acid sequence of OsGSTU41.

SEQ ID NO: 6 is the nucleotide sequence of cDNA of OsPPCK4 gene.

SEQ ID NO: 7 is the nucleotide sequence of CDS of OsPPCK4 gene.

SEQ ID NO: 8 is the amino acid sequence of OsPPCK4.

SEQ ID NO: 9 is the nucleotide sequence of cDNA of OsCAM2 gene.

SEQ ID NO: 10 is the nucleotide sequence of CDS of OsCAM2 gene.

SEQ ID NO: 11 is the amino acid sequence of OsCAM2.

SEQ ID NO: 12 is the nucleotide sequence of gDNA of OsDN-DTP4 gene.

SEQ ID NO: 13 is the nucleotide sequence of CDS of OsDN-DTP4 gene.

SEQ ID NO: 14 is the amino acid sequence of OsDN-DTP4.

SEQ ID NO: 15 is the nucleotide sequence of gDNA of OsLecRK4.1 gene.

SEQ ID NO: 16 is the nucleotide sequence of CDS of OsLecRK4.1 gene.

SEQ ID NO: 17 is the amino acid sequence of OsLecRK4.1.

SEQ ID NO: 18 is the nucleotide sequence of gDNA of OsLecRK4.2 gene.

SEQ ID NO: 19 is the nucleotide sequence of CDS of OsLecRK4.2 gene.

SEQ ID NO: 20 is the amino acid sequence of OsLecRK4.2.

SEQ ID NO: 21 is forward primer for cloning cDNA of OsGSTU41 gene.

SEQ ID NO: 22 is reverse primer for cloning cDNA of OsGSTU41 gene.

SEQ ID NO: 23 is forward primer for cloning cDNA of OsPPCK4 gene.

SEQ ID NO: 24 is reverse primer for cloning cDNA of OsPPCK4 gene.

SEQ ID NO: 25 is forward primer for cloning cDNA of OsCAM2 gene.

SEQ ID NO: 26 is reverse primer for cloning cDNA of OsCAM2 gene.

SEQ ID NO: 27 is forward primer for cloning gDNA of OsDN-DTP4 gene.

SEQ ID NO: 28 is reverse primer for cloning gDNA of OsDN-DTP4 gene.

SEQ ID NO: 29 is forward primer for cloning gDNA of OsLecRK4.1 gene.

SEQ ID NO: 30 is reverse primer for cloning gDNA of OsLecRK4.1 gene.

SEQ ID NO: 31 is forward primer for cloning gDNA of OsLecRK4.2 gene.

SEQ ID NO: 32 is reverse primer for cloning gDNA of OsLecRK4.2 gene.

SEQ ID NO: 33 is forward primer for real-time RT-PCR analysis of OsGSTU41 gene.

SEQ ID NO: 34 is reverse primer for real-time RT-PCR analysis of OsGSTU41 gene.

SEQ ID NO: 35 is forward primer for real-time RT-PCR analysis of OsPPCK4 gene.

SEQ ID NO: 36 is reverse primer for real-time RT-PCR analysis of OsPPCK4 gene.

SEQ ID NO: 37 is forward primer for real-time RT-PCR analysis of OsDN-DTP4 gene.

SEQ ID NO: 38 is reverse primer for real-time RT-PCR analysis of OsDN-DTP4 gene.

SEQ ID NO: 39 is forward primer for real-time RT-PCR analysis of OsLecRK4.1 gene.

SEQ ID NO: 40 is reverse primer for real-time RT-PCR analysis of OsLecRK4.1 gene.

SEQ ID NO: 41 is forward primer for real-time RT-PCR analysis of OsLecRK4.2 gene.

SEQ ID NO: 42 is reverse primer for real-time RT-PCR analysis of OsLecRK4.2 gene.

DETAILED DESCRIPTION

The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants; reference to “a cell” includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.

As used herein:

The term “OsGSTU41 (glutathione S-transferase TAU41)” refers to a rice polypeptide that confers drought tolerance and paraquat tolerance phenotype and is encoded by the rice gene locus LOC_Os01g72160.1. “GSTU41 polypeptide” refers herein to the OsGSTU41 polypeptide and its homologs from other organisms.

The OsGSTU41 polypeptide (SEQ ID NO: 5) is encoded by the coding sequence (CDS) (SEQ ID NO: 4) or nucleotide sequence (SEQ ID NO: 3) at rice gene locus LOC_Os01g72160.1. This polypeptide is annotated as “glutathione S-transferase, putative, expressed” in TIGR.

The term “OsPPCK4 (phosphoenolpyruvate carboxylase kinase 4)” refers to a rice polypeptide that confers drought tolerance and paraquat tolerance phenotype and is encoded by the rice gene locus LOC_Os02g56310.1. “PPCK4 polypeptide” refers herein to the OsPPCK4 polypeptide and its homologs from other organisms.

The OsPPCK4 polypeptide (SEQ ID NO: 8) is encoded by the coding sequence (CDS) (SEQ ID NO: 7) or nucleotide sequence (SEQ ID NO: 6) at rice gene locus LOC_Os02g56310.1. This polypeptide is annotated as “calcium-dependent protein kinase isoform AK1, putative, expressed” in TIGR and “putative phosphoenolpyruvate carboxylase kinase” in NCBI (on the world web at ncbi.nlm.nih.gov).

The term “OsCAM2 (Calmodulin 2)” refers to a rice polypeptide that confers drought tolerance and paraquat tolerance and is encoded by the rice gene locus LOC_Os05g41210.1. “CAM2 polypeptide” refers herein to the OsCAM2 polypeptide and its homologs from other organisms.

The OsCAM2 polypeptide (SEQ ID NO: 11) is encoded by the coding sequence (CDS) (SEQ ID NO: 10) or nucleotide sequence (SEQ ID NO: 9) at rice gene locus LOC_Os05g41210.1. This polypeptide is annotated as “Oscam2-Calmodulin, expressed” in TIGR.

The term “OsDN-DTP4 (drought tolerance protein 4)” refers to a rice polypeptide that confers drought tolerance and pataquat tolerance phenotype and is encoded by the rice gene locus LOC_Os07g04720.1. “DN-DTP4 polypeptide” refers herein to the OsDN-DTP4 polypeptide and its homologs from other organisms.

The OsDN-DTP4 polypeptide (SEQ ID NO: 14) is encoded by the coding sequence (CDS) (SEQ ID NO: 13) or nucleotide sequence (SEQ ID NO: 12) at rice gene locus LOC_Os07g04720.1. This polypeptide is annotated as “expressed protein” in TIGR.

The term “OsLecRK4.1 (lectin-like receptor kinase 4.1)” refers to a rice polypeptide that confers drought tolerance and paraquat tolerance and is encoded by the rice gene locus LOC_Os04g44900.1. “LecRK4.1 polypeptide” refers herein to the OsLecRK4.1 polypeptide and its homologs from other organisms.

The OsLecRK4.1 polypeptide (SEQ ID NO: 17) is encoded by the coding sequence (CDS) (SEQ ID NO: 16) or nucleotide sequence (SEQ ID NO: 15) at rice gene locus LOC_Os04g44900.1. This polypeptide is annotated as “lectin-like receptor kinase, putative, expressed” in TIGR.

The term “OsLecRK4.2 (lectin-like receptor kinase 4.2)” refers to a rice polypeptide that confers drought and paraquat tolerance and is encoded by the rice gene locus LOC_Os04g44910.1. “LecRK4.2 polypeptide” refers herein to the OsLecRK4.2 polypeptide and its homologs from other organisms.

The OsLecRK4.2 polypeptide (SEQ ID NO: 20) is encoded by the coding sequence (CDS) (SEQ ID NO: 19) or nucleotide sequence (SEQ ID NO: 18) at rice gene locus LOC_Os04g44910.1. This polypeptide is annotated as “receptor like protein kinase, putative, expressed” in TIGR.

The terms “monocot” and “monocotyledonous plant” are used interchangeably herein. A monocot of the current disclosure includes plants of the Gramineae family.

The terms “dicot” and “dicotyledonous plant” are used interchangeably herein. A dicot of the current disclosure includes the following families: Brassicaceae, Leguminosae, and Solanaceae.

The terms “full complement” and “full-length complement” are used interchangeably herein, and refer to a complement of a given nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from a cDNA library and therefore represents a sequence which has been transcribed. An EST is typically obtained by a single sequencing pass of a cDNA insert. The sequence of an entire cDNA insert is termed the “Full-Insert Sequence” (“FIS”). A “Contig” sequence is a sequence assembled from two or more sequences that can be selected from, but not limited to, the group consisting of an EST, FIS and PCR sequence. A sequence encoding an entire or functional protein is termed a “Complete Gene Sequence” (“CGS”) and can be derived from an FIS or a contig.

The term “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar or nitrogen concentrations, or by the observation of the expression level of a gene or genes, or by agricultural observations such as osmotic stress tolerance or yield.

“Agronomic characteristic” is a measurable parameter including but not limited to: greenness, grain yield, growth rate, total biomass or rate of accumulation, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, salt tolerance, tiller number, panicle size, early seedling vigor and seedling emergence under low temperature stress.

Increased biomass can be measured, for example, as an increase in plant height, plant total leaf area, plant fresh weight, plant dry weight or plant seed yield, as compared with control plants.

The ability to increase the biomass or size of a plant would have several important commercial applications. Crop cultivars may be developed to produce higher yield of the vegetative portion of the plant, to be used in food, feed, fiber, and/or biofuel.

Increased leaf size may be of particular interest. Increased leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. Increased tiller number may be of particular interest and can be used to increase yield. An increase in total plant photosynthesis is typically achieved by increasing leaf area of the plant. Additional photosynthetic capacity may be used to increase the yield derived from particular plant tissue, including the leaves, roots, fruits or seed, or permit the growth of a plant under decreased light intensity or under high light intensity.

Modification of the biomass of another tissue, such as root tissue, may be useful to improve a plant's ability to grow under harsh environmental conditions, including drought or nutrient deprivation, because larger roots may better reach or take up water or nutrients.

For some ornamental plants, the ability to provide larger varieties would be highly desirable. For many plants, including fruit-bearing trees, trees that are used for lumber production, or trees and shrubs that serve as view or wind screens, increased stature provides improved benefits, such as in the forms of greater yield or improved screening.

“Transgenic” refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event. The term “transgenic” used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

A “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of a subject plant or plant cell in which genetic alteration, such as transformation, has been effected as to a gene of interest. A subject plant or plant cell may be descended from a plant or cell so altered and will comprise the alteration.

A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to a condition or stimulus that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

“Genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but also organelle DNA found within subcellular components (e.g., mitochondria, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissues, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises within its genome a heterologous polynucleotide. For example, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. A T₀ plant is directly recovered from the transformation and regeneration process. Progeny of T₀ plants are referred to as T₁ (first progeny generation), T₂ (second progeny generation), etc.

“Heterologous” with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, and “nucleic acid fragment” are used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single-letter designation as follows: “A” for adenylate or deoxyadenylate, “C” for cytidylate or deoxycytidylate, and “G” for guanylate or deoxyguanylate for RNA or DNA, respectively; “U” for uridylate; “T” for deoxythymidylate; “R” for purines (A or G); “Y” for pyrimidines (C or T); “K” for G or T; “H” for A or C or T; “I” for inosine; and “N” for any nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, and sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA which has no intron and can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from an mRNA template using reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.

“Mature” protein refers to a post-translation processed polypeptide; i.e., any pre- or pro-peptides present in the primary translation product has been removed.

“Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.

“Isolated” refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. “Recombinant” also includes reference to a cell or vector, that has been modified by the introduction of a heterogonous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.

The terms “entry clone” and “entry vector” are used interchangeably herein.

“Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and influencing the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and poly-adenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controlling transcription of genes in plant cells whether or not its origin is from a plant cell.

“Tissue-specific promoter” and “tissue-preferred promoter” may refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell or cell type.

“Developmentally regulated promoter” refers to a promoter whose activity is determined by developmental events.

“Operably linked” refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.

“Expression” refers to the production of a functional product. For example, expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.

“Phenotype” means the detectable characteristic or characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation and transient transformation.

“Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.

“Transient transformation” refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.

An “allele” is one of two or more alternative forms of a gene occupying a given locus on a chromosome. When the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant are the same, that plant is homozygous at that locus. If the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant differ, that plant is heterozygous at that locus. If a transgene is present on one of a pair of homologous chromosomes in a diploid plant, that plant is hemizygous at that locus.

A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels. (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel. (1992) Plant Phys. 100:1627-1632). A “mitochondrial signal peptide” is an amino acid sequence which directs a precursor protein into the mitochondria (Zhang and Glaser. (2002) Trends Plant Sci 7:14-21).

Methods to determine the relationship of various polynucleotide and polypeptide sequences are known. As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence, such as a segment of a full-length cDNA or gene sequence, or may be the complete cDNA or gene sequence. As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide or polypeptide sequence, wherein the sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides or amino acids in length, and optionally can be 30, 40, 50, 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the sequence, a gap penalty is typically introduced and is subtracted from the number of matches.

The determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Examples of such mathematical algorithms for sequence comparison include the algorithm of Myers and Miller. (1988) CABIOS 4:11-17; the local alignment algorithm of Smith, et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch. (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman. (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul. (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul. (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA); and the Megalign® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.).

Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins, et al. (1988) Gene 73:237-244; Higgins, et al. (1989) CABIOS 5:151-153; Corpet, et al. (1988) Nucleic Acids Res. 16:10881-10890; Huang, et al. (1992) CABIOS 8:155-165 and Pearson, et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller, (1988) supra. A PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul. (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the disclosures. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the disclosures. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules (Altschul, et al. (1997) supra). When utilizing BLAST, Gapped BLAST, PSI-BLAST and the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used (the National Center for Biotechnology Information of the National Library of Medicine of the National Institutes of Health of the U.S. government). Alignment may also be performed by manual inspection.

Paired sequence identity/similarity values can be obtained using GAP Version 10 with the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch. (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the Quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (Henikoff and Henikoff. (1989) Proc. Natl. Acad. Sci. USA 89:10915).

Unless stated otherwise, multiple alignments of the sequences provided herein are performed using the Clustal V method of alignment (Higgins and Sharp. (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of amino acid sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.

As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).

Embodiments include isolated polynucleotides and polypeptides, and recombinant DNA constructs useful for conferring drought tolerance; compositions (such as plants or seeds) comprising these recombinant DNA constructs; and methods utilizing these recombinant DNA constructs.

Isolated Polynucleotides and Polypeptides:

The present disclosure includes the following isolated polynucleotides and polypeptides:

An isolated polynucleotide comprising: (i) a nucleic acid sequence encoding a polypeptide having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20; or (ii) a full complement of the nucleic acid sequence of (i), wherein the full complement and the nucleic acid sequence of (i) consist of the same number of nucleotides and are 100% complementary. Any of the foregoing isolated polynucleotides may be utilized in any recombinant DNA constructs of the present disclosure. Over-expression of the encoded polypeptide increases drought tolerance, and/or paraquat tolerance activity in plant.

An isolated polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 6.4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20. The polypeptide is preferably a drought tolerance polypeptide. Over-expression of the polypeptide increases drought tolerance, and/or paraquat tolerance activity in plant.

An isolated polynucleotide comprising (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 5.4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 6.4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, 7, 10, 13, 16 or 19; (ii) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 6.4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, 6, 9, 12, 15 or 18; or (iii) a full complement of the nucleic acid sequence of (i) or (ii). Any of the foregoing isolated polynucleotides may be utilized in any recombinant DNA constructs of the present disclosure. The isolated polynucleotide preferably encodes a drought tolerance polypeptide. Over-expression of the polypeptide improves drought tolerance and/or paraquat tolerance activity in plant.

Recombinant DNA Constructs:

In one aspect, the present disclosure includes recombinant DNA constructs.

In one embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein the polynucleotide comprises (i) a nucleic acid sequence encoding an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20; or (ii) a full complement of the nucleic acid sequence of (i).

In another embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein said polynucleotide comprises (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, 7, 10, 13, 16 or 19; (ii) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, 6, 9, 12, 15 or 18; or (iii) a full complement of the nucleic acid sequence of (i) or (ii).

In another embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein said polynucleotide encodes a drought tolerance polypeptide. The polypeptide preferably has drought tolerance and/or paraquat tolerance activity. The polypeptide may be from, for example, Oryza sativa, Oryza australiensis, Oryza barthii, Oryza glaberrima (African rice), Oryza latifolia, Oryza longistaminata, Oryza meridionalis, Oryza officinalis, Oryza punctata, Oryza rufipogon (brownbeard or red rice), Oryza nivara (Indian wild rice), Arabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja or Glycine tomentella.

It is understood, as those skilled in the art will appreciate, that the disclosure encompasses more than the specific exemplary sequences. Alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

“Suppression DNA construct” is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in “silencing” of a target gene in the plant. The target gene may be endogenous or transgenic to the plant. “Silencing,” as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality. The terms “suppression”, “suppressing” and “silencing”, used interchangeably herein, include lowering, reducing, declining, decreasing, inhibiting, eliminating or preventing. “Silencing” or “gene silencing” does not specify mechanism and is inclusive of, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending upon the approach to be utilized, the region may be 100% identical or less than 100% identical (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sense strand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target gene or gene product. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (for example, U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with respect to any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcripts capable of suppressing the expression of the target gene or gene product. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on over-expression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the over-expressed sequence (Vaucheret et al. (1998) Plant J. 16:651-659; and Gura. (2000) Nature 404:804-808).

RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing (PTGS) in animals mediated by short interfering RNAs (siRNAs) (Fire et al. (1998) Nature 391:806). The corresponding process in plants is commonly referred to as PTGS or RNA silencing and is also referred to as quelling in fungi. The process of PTGS is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al. (1999) Trends Genet. 15:358).

Small RNAs play an important role in controlling gene expression, for example, small RNAs regulate many developmental processes which include flowering. It is now possible to engineer changes in gene expression of plant genes by using transgenic constructs which produce small RNAs in the plant.

Small RNAs appear to function by base-pairing to complementary RNA or DNA target sequences. When bound to RNA, small RNAs trigger either RNA cleavage or translational inhibition of the target sequence. When bound to DNA target sequences, it is thought that small RNAs can mediate DNA methylation of the target sequence. The consequence of these events, regardless of the specific mechanism, is that gene expression is inhibited.

MicroRNAs (miRNAs) are noncoding RNAs of about 19 to 24 nucleotides (nt) in length that have been identified in both animals and plants (Lagos-Quintana et al. (2001) Science 294:853-858, Lagos-Quintana et al. (2002) Curr. Biol. 12:735-739; Lau et al. (2001) Science 294:858-862; Lee and Ambros. (2001) Science 294:862-864; Llave et al. (2002) Plant Cell 14:1605-1619; Mourelatos et al. (2002) Genes Dev. 16:720-728; Park et al. (2002)Curr. Biol. 12:1484-1495; Reinhart et al. (2002) Genes Dev. 16: 1616-1626). They are processed from longer precursor transcripts that range in size from approximately 70 to 200 nt, and these precursor transcripts have the ability to form stable hairpin structures.

miRNAs appear to regulate target genes by binding to complementary sequences located in the transcripts produced by these genes. It seems likely that miRNAs can enter at least two pathways of target gene regulation: (1) translational inhibition; and (2) RNA cleavage. miRNAs entering the RNA cleavage pathway are analogous to the 21-25 nt siRNAs generated during RNAi in animals and PTGS in plants, and likely are incorporated into an RNA-induced silencing complex (RISC) that is similar or identical to that seen for RNAi.

Regulatory Sequences:

A recombinant DNA construct of the present disclosure may comprise at least one regulatory sequence.

A regulatory sequence may be a promoter.

A number of promoters can be used in recombinant DNA constructs of the present disclosure. The promoters can be selected based on the desired outcome, and may include constitutive, tissue-specific, inducible, or other promoters for expression in the host organism.

Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.

High-level, constitutive expression of the candidate gene under control of the 35S or UBI promoter may have pleiotropic effects, although candidate gene efficacy may be estimated when driven by a constitutive promoter. Use of tissue-specific and/or stress-induced promoters may eliminate undesirable effects but retain the ability to enhance drought tolerance. This effect has been observed in Arabidopsis (Kasuga et al. (1999) Nature Biotechnol. 17:287-91).

Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

In choosing a promoter to use in the methods of the disclosure, it may be desirable to use a tissue-specific or developmentally regulated promoter.

A tissue-specific or developmentally-regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in the cells/tissues of a plant, such as in those cells/tissues critical to tassel development, seed set, or both, and which usually limits the expression of such a DNA sequence to the developmental period of interest (e.g. tassel development or seed maturation) in the plant. Any identifiable promoter which causes the desired temporal and spatial expression may be used in the methods of the present disclosure.

Many leaf-preferred promoters are known in the art (Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-367; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-518; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590).

Promoters which are seed or embryo-specific and may be useful in the disclosure include soybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg. (1989) Plant Cell 1:1079-1093), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W. G., et al. (1991) Mol. Gen. Genet. 259:149-157; Newbigin, E. J., et al. (1990) Planta 180:461-470; Higgins, T. J. V., et al. (1988) Plant. Mol. Biol. 11:683-695), zein (maize endosperm) (Schemthaner, J. P., et al. (1988) EMBO J. 7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al. (1985) Proc. Natl. Acad. Sci. 82:3320-3324), phytohemagglutinin (bean cotyledon) (Voelker, T. et al. (1987) EMBO J. 6:3571-3577), B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, et al. (1988) EMBO J. 7:297-302), glutelin (rice endosperm), hordein (barley endosperm) (Marris, C., et al. (1988) Plant Mol. Biol. 10:359-366), glutenin and gliadin (wheat endosperm) (Colot, V., et al. (1987) EMBO J. 6:3559-3564). Promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructions maintain their temporal and spatial expression pattern in transgenic plants. Such examples include Arabidopsis 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds (Vanderkerckhove et al. (1989) Bio/Technology 7:L929-932), bean lectin and bean beta-phaseolin promoters to express luciferase (Riggs et al. (1989) Plant Sci. 63:47-57), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al. (1987) EMBO J 6:3559-3564).

Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.

Promoters for use in certain embodiments include the following: 1) the stress-inducible promoter RD29A (Kasuga et al. (1999) Nature Biotechnol. 17:287-291); 2) the stress-inducible promoter Rab17 (Vilardell et al. (1991) Plant Mol. Bio. 17:985-993; Kamp Busk et al. (1997) Plant J 11(6):1285-1295); 3) the barley promoter B22E whose expression is specific to the pedicel in developing maize kernels (“Primary Structure of a Novel Barley Gene Differentially Expressed in Immature Aleurone Layers”. Klemsdal, S. S. et al. (1991) Mol. Gen. Genet. 228(1/2):9-16); and 4) maize promoter Zag2 (“Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS”, Schmidt, R. J. et al. (1993) Plant Cell 5(7):729-737; “Structural characterization, chromosomal localization and phylogenetic evaluation of two pairs of AGAMOUS-like MADS-box genes from maize”, Theissen et al. (1995) Gene 156(2):155-166; NCBI GenBank Accession No. X80206)). Zag2 transcripts can be detected 5 days prior to pollination to 7 to 8 days after pollination (“DAP”), and directs expression in the carpel of developing female inflorescences and Ciml which is specific to the nucleus of developing maize kernels. Ciml transcript is detected 4 to 5 days before pollination to 6 to 8 DAP. Other useful promoters include any promoter which can be derived from a gene whose expression is maternally associated with developing female florets.

For the expression of a polynucleotide in developing seed tissue, promoters of particular interest include seed-preferred promoters, particularly early kernel/embryo promoters and late kernel/embryo promoters. Kernel development post-pollination is divided into approximately three primary phases. The lag phase of kernel growth occurs from about 0 to 10-12 DAP. During this phase the kernel is not growing significantly in mass, but rather important events are being carried out that will determine kernel vitality (e.g., number of cells established). The linear grain fill stage begins at about 10-12 DAP and continues to about 40 DAP. During this stage of kernel development, the kernel attains almost all of its final mass, and various storage products (i.e., starch, protein, oil) are produced. Finally, the maturation phase occurs from about 40 DAP to harvest. During this phase of kernel development the kernel becomes quiescent and begins to dry down in preparation for a long period of dormancy prior to germination. As defined herein “early kernel/embryo promoters” are promoters that drive expression principally in developing seed during the lag phase of development (i.e., from about 0 to about 12 DAP). “Late kernel/embryo promoters”, as defined herein, drive expression principally in developing seed from about 12 DAP through maturation. There may be some overlap in the window of expression. The choice of the promoter will depend on the ABA-associated sequence utilized and the phenotype desired.

Early kernel/embryo promoters include, for example, Cim1 that is active 5 DAP in particular tissues (WO 00/11177), which is herein incorporated by reference. Other early kernel/embryo promoters include the seed-preferred promoters end1 which is active 7-10 DAP, and end2, which is active 9-14 DAP in the whole kernel and active 10 DAP in the endosperm and pericarp (WO 00/12733), herein incorporated by reference. Additional early kernel/embryo promoters that find use in certain methods of the present disclosure include the seed-preferred promoter Itp2 (U.S. Pat. No. 5,525,716); maize Zm40 promoter (U.S. Pat. No. 6,403,862); maize nuc1c (U.S. Pat. No. 6,407,315); maize ckx1-2 promoter (U.S. Pat. No. 6,921,815 and US Patent Application Publication Number 2006/0037103); maize led promoter (U.S. Pat. No. 7,122,658); maize ESR promoter (U.S. Pat. No. 7,276,596); maize ZAP promoter (U.S. Patent Application Publication Numbers 20040025206 and 20070136891); maize promoter eep1 (U.S. Patent Application Publication Number 20070169226); and maize promoter ADF4 (U.S. Patent Application No. 60/963,878, filed 7 Aug. 2007).

Additional promoters for regulating the expression of the nucleotide sequences of the present disclosure in plants are stalk-specific promoters, including the alfalfa S2A promoter (GenBank Accession No. EF030816; Abrahams et al. (1995) Plant Mol. Biol. 27:513-528) and S2B promoter (GenBank Accession No. EF030817) and the like, herein incorporated by reference.

Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.

Promoters for use in certain embodiments of the current disclosure may include: RIP2, mLIP15, ZmCOR1, Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM synthetase, ubiquitin, CaMV 19S, nos, Adh, sucrose synthase, R-allele, the vascular tissue preferred promoters S2A (Genbank accession number EF030816) and S2B (Genbank accession number EF030817), and the constitutive promoter GOS2 from Zea mays; root preferred promoters, such as the maize NAS2 promoter, the maize Cyclo promoter (US 2006/0156439, published Jul. 13, 2006), the maize ROOTMET2 promoter (WO05063998, published Jul. 14, 2005), the CR1 BIO promoter (WO06055487, published May 26, 2006), the CRWAQ81 (WO05035770, published Apr. 21, 2005) and the maize ZRP2.47 promoter (NCBI accession number: U38790; GI No. 1063664).

Recombinant DNA constructs of the present disclosure may also include other regulatory sequences, including but not limited to, translation leader sequences, introns, and polyadenylation recognition sequences. In certain embodiments, a recombinant DNA construct further comprises an enhancer or silencer.

An intron sequence can be added to the 5′ untranslated region, the protein-coding region or the 3′ untranslated region to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg. (1988) Mol. Cell Biol. 8:4395-4405; Callis et al. (1987) Genes Dev. 1:1183-1200).

Any plant can be selected for the identification of regulatory sequences and polypeptide genes to be used in recombinant DNA constructs of the present disclosure. Examples of suitable plant targets for the isolation of genes and regulatory sequences would include but are not limited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, ornamental plant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, switchgrass, tangerine, tea, tobacco, tomato, triticale, turf, turnip, vine, watermelon, wheat, yams, and zucchini.

Compositions:

A composition of the present disclosure is a plant comprising in its genome any of the recombinant DNA constructs of the present disclosure (such as any of the constructs discussed above). Compositions also include any progeny of the plant, and any seed obtained from the plant or its progeny, wherein the progeny or seed comprises within its genome the recombinant DNA construct. Progeny includes subsequent generations obtained by self-pollination or out-crossing of a plant. Progeny also includes hybrids and inbreds.

In hybrid seed propagated crops, mature transgenic plants can be self-pollinated to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced recombinant DNA construct. These seeds can be grown to produce plants that would exhibit an altered agronomic characteristic (e.g., an increased agronomic characteristic optionally under water limiting conditions), or used in a breeding program to produce hybrid seed, which can be grown to produce plants that would exhibit such an altered agronomic characteristic. The seeds may be maize seeds or rice seeds.

The plant may be a monocotyledonous or dicotyledonous plant, for example, a rice or maize or soybean plant, such as a maize hybrid plant or a maize inbred plant. The plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, barley, millet, sugar cane or switchgrass.

The recombinant DNA construct may be stably integrated into the genome of the plant.

Particular embodiments include but are not limited to the following:

1. A transgenic plant (for example, a rice or maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20, and wherein said plant exhibits increased drought tolerance and/or paraquat tolerance when compared to a control plant. The plant may further exhibit an alteration of at least one agronomic characteristic when compared to the control plant.

2. A transgenic plant (for example, a rice or maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a drought tolerance polypeptide, and wherein said plant exhibits increased drought tolerance and/or paraquat tolerance when compared to a control plant. The plant may further exhibit an alteration of at least one agronomic characteristic when compared to the control plant.

3. A transgenic plant (for example, a rice or maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a drought tolerance polypeptide, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant.

4. Any progeny of the above plants in embodiment 1-3, any seeds of the above plants in embodiment 1-3, any seeds of progeny of the above plants in embodiment 1-3, and cells from any of the above plants in embodiment 1-3 and progeny thereof.

In any of the foregoing embodiment 1-4 or other embodiments, the drought tolerance polypeptide may be from Oryza sativa, Oryza australiensis, Oryza barthii, Oryza glaberrima (African rice), Oryza latifolia, Oryza longistaminata, Oryza meridionalis, Oryza officinalis, Oryza punctata, Oryza rufipogon (brownbeard or red rice), Oryza nivara (Indian wild rice), Arabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja or Glycine tomentella.

In any of the foregoing embodiment 1-4 or other embodiments, the recombinant DNA construct may comprise at least a promoter functional in a plant as a regulatory sequence.

In any of the foregoing embodiment 1-4 or other embodiments, the alteration of at least one agronomic characteristic is either an increase or decrease.

In any of the foregoing embodiment 1-4 or other embodiments, the at least one agronomic characteristic may be selected from the group consisting of greenness, grain yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, salt tolerance, tiller number, panicle size, early seedling vigor and seedling emergence under low temperature stress. For example, the alteration of at least one agronomic characteristic may be an increase in grain yield, greenness or biomass.

In any of the foregoing embodiment 1-4 or other embodiments, the plant may exhibit the alteration of at least one agronomic characteristic when compared, under water limiting conditions, to a control plant.

In any of the foregoing embodiment 1-4 or other embodiments, the plant may exhibit the alteration of at least one agronomic characteristic when compared, under oxidative stress (paraquat) conditions, to a control plant.

“Drought” refers to a decrease in water availability to a plant that, especially when prolonged or when occurring during critical growth periods, can cause damage to the plant or prevent its successful growth (e.g., limiting plant growth or seed yield).

“Drought tolerance” reflects a plant's ability to survive under drought without exhibiting substantial physiological or physical deterioration, and/or its ability to recover when water is restored following a period of drought.

“Drought tolerance activity” of a polypeptide indicates that over-expression of the polypeptide in a transgenic plant confers increased drought tolerance of the transgenic plant relative to a reference or control plant.

“Increased drought tolerance” of a plant is measured relative to a reference or control plant, and reflects ability of the plant to survive under drought conditions with less physiological or physical deterioration than a reference or control plant grown under similar drought conditions, or ability of the plant to recover more substantially and/or more quickly than would a control plant when water is restored following a period of drought.

“Environmental conditions” refer to conditions under which the plant is grown, such as the availability of water, availability of nutrients, or the presence of insects or disease.

“Paraquat” (1,1-dimethyl-4,4-bipyridinium dichloride), is a foliar-applied and non-selective bipyridinium herbicides, and causes photooxidative stress which further cause damage to plant or prevent its successful growth.

“Paraquat tolerance” is a trait of a plant, reflects the ability to survive and/or grow better when treated with Paraquat solution, compared to a reference or control plant.

“Increased paraquat tolerance” of a plant is measured relative to a reference or control plant, and reflects ability of the plant to survive with less physiological or physical deterioration than a reference or control plant after treated with paraquat solution. In general, tolerance to relative low level of paraquat can be used as a marker of abiotic stress tolerance, such as drought tolerance.

“Oxidative stress” reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA.

The Examples below describe some representative protocols and techniques for simulating drought conditions and/or evaluating drought tolerance; and simulating oxidative conditions.

One can also evaluate drought tolerance by the ability of a plant to maintain sufficient yield (at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% yield) in field testing under simulated or naturally-occurring drought conditions (e.g., by measuring for substantially equivalent yield under drought conditions compared to non-drought conditions, or by measuring for less yield loss under drought conditions compared to yield loss exhibited by a control or reference plant).

Parameters such as recovery degree, survival rate, paraquat tolerance rate, gene expression level, water use efficiency, level or activity of an encoded protein, and others are typically presented with reference to a control cell or control plant. A “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of a subject plant or plant cell in which genetic alteration, such as transformation, has been effected as to a gene of interest. A subject plant or plant cell may be descended from a plant or cell so altered and will comprise the alteration. One of ordinary skill in the art would readily recognize a suitable control or reference plant to be utilized when assessing or measuring an agronomic characteristic or phenotype of a transgenic plant using compositions or methods as described herein. For example, by way of non-limiting illustrations:

1. Progeny of a transformed plant which is hemizygous with respect to a recombinant DNA construct, such that the progeny are segregating into plants either comprising or not comprising the recombinant DNA construct: the progeny comprising the recombinant DNA construct would be typically measured relative to the progeny not comprising the recombinant DNA construct. The progeny not comprising the recombinant DNA construct is the control or reference plant.

2. Introgression of a recombinant DNA construct into an inbred line, such as in rice and maize, or into a variety, such as in soybean: the introgressed line would typically be measured relative to the parent inbred or variety line (i.e., the parent inbred or variety line is the control or reference plant).

3. Two hybrid lines, wherein the first hybrid line is produced from two parent inbred lines, and the second hybrid line is produced from the same two parent inbred lines except that one of the parent inbred lines contains a recombinant DNA construct: the second hybrid line would typically be measured relative to the first hybrid line (i.e., the first hybrid line is the control or reference plant).

4. A plant comprising a recombinant DNA construct: the plant may be assessed or measured relative to a control plant not comprising the recombinant DNA construct but otherwise having a comparable genetic background to the plant (e.g., sharing at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of nuclear genetic material compared to the plant comprising the recombinant DNA construct. There are many laboratory-based techniques available for the analysis, comparison and characterization of plant genetic backgrounds; among these are Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLP®s), and Simple Sequence Repeats (SSRs) which are also referred to as Microsatellites.

A control plant or plant cell may comprise, for example: (a) a wild-type (WT) plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimulus that would induce expression of the gene of interest or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed. A control may comprise numerous individuals representing one or more of the categories above; for example, a collection of the non-transformed segregants of category “c” is often referred to as a bulk null.

In this disclosure, Line null, Bulk null, ZH11-TC and VC indicate control plants, Line null represents the control segregated from the corresponding hemizygous transgenic lines, Bulk null represents the bulk null which is a collection of the non-transformed segregants from the hemizygous transgenic lines, ZH11-TC represents rice plants generated from tissue cultured Zhonghua 11, and VC represents plants transformed with empty vector of DP0005 or DP0158.

Methods:

Methods include but are not limited to methods for increasing drought tolerance in a plant, methods for evaluating drought tolerance in a plant, methods for increasing paraquat tolerance, methods for altering an agronomic characteristic in a plant, methods for determining an alteration of an agronomic characteristic in a plant, and methods for producing seed. The plant may be a monocotyledonous or dicotyledonous plant, for example, rice, maize or soybean plant. The plant may also be sunflower, canola, wheat, alfalfa, cotton, barley, millet, sugar cane or sorghum. The seed may be a maize or soybean seed, for example, a maize hybrid seed or maize inbred seed.

Methods include but not limited to the following:

A method for transforming a cell comprising transforming a cell with any one or more of the isolated polynucleotides of the present disclosure, wherein, in particular embodiments, the cell is eukaryotic cell, e.g., a yeast, insect or plant cell; or prokaryotic cell, e.g., a bacterial cell.

A method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides or recombinant DNA constructs of the present disclosure and regenerating a transgenic plant from the transformed plant cell, wherein, the transgenic plant and the transgenic seed obtained by this method may be used in other methods of the present disclosure.

A method for isolating a polypeptide of the disclosure from a cell or culture medium of the cell, wherein the cell comprises a recombinant DNA construct comprising a polynucleotide of the disclosure operably linked to at least one regulatory sequence, and wherein the transformed host cell is grown under conditions that are suitable for expression of the recombinant DNA construct.

A method for altering the level of expression of a polypeptide of the disclosure in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present disclosure; and (b) growing the transformed host cell under conditions that are suitable for the expression of the recombinant DNA construct, wherein the expression of the recombinant DNA construct results in production of altered levels of the polypeptide of the disclosure in the transformed host cell.

A method of increasing drought tolerance and/or paraquat tolerance in a plant, comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits increased drought tolerance and/or paraquat tolerance when compared to a control plant; and further (c) obtaining a progeny plant derived from transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits increased drought tolerance and/or paraquat tolerance when compared to a control plant.

A method of evaluating drought tolerance and/or paraquat tolerance in a plant comprising (a) obtaining a transgenic plant, which comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, 8, 11, 14, 17 or 20; (b) obtaining a progeny plant derived from said transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (c) evaluating the progeny plant for drought tolerance and/or paraquat tolerance compared to a control plant.

A method of determining an alteration of an agronomic characteristic in a plant comprising (a) obtaining a transgenic plant which comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity when compared to SEQ ID NO: 5, 8, 11, 14, 17 or 20; (b) obtaining a progeny plant derived from said transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (c) determining whether the progeny plant exhibits an alteration in at least one agronomic characteristic when compared, optionally under water limiting conditions, to a control plant.

A method of producing seed comprising any of the preceding methods, and further comprising obtaining seeds from said progeny plant, wherein said seeds comprise in their genome said recombinant DNA construct.

In any of the preceding methods or any other embodiments of methods of the present disclosure, in said introducing step, the said regenerable plant cell may comprise a callus cell, an embryogenic callus cell, a gametic cell, a meristematic cell, or a cell of an immature embryo. The regenerable plant cells may derive from an inbred maize plant.

In any of the preceding methods or any other embodiments of methods of the present disclosure, said regenerating step may comprise the following: (i) culturing said transformed plant cells in a medium comprising an embryogenic promoting hormone until callus organization is observed; (ii) transferring said transformed plant cells of step (i) to a first media which includes a tissue organization promoting hormone; and (iii) subculturing said transformed plant cells after step (ii) onto a second media, to allow for shoot elongation, root development or both.

In any of the preceding methods or any other embodiments of methods of the present disclosure, the step of determining an alteration of an agronomic characteristic in a transgenic plant, if applicable, may comprise determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.

In any of the preceding methods or any other embodiments of methods of the present disclosure, the step of determining an alteration of an agronomic characteristic in a progeny plant, if applicable, may comprise determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.

In any of the preceding methods or any other embodiments of methods of the present disclosure, the plant may exhibit the alteration of at least one agronomic characteristic when compared, under water limiting conditions, to a control plant.

In any of the preceding methods or any other embodiments of methods of the present disclosure, alternatives exist for introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence. For example, one may introduce into a regenerable plant cell a regulatory sequence (such as one or more enhancers, optionally as part of a transposable element), and then screen for an event in which the regulatory sequence is operably linked to an endogenous gene encoding a polypeptide of the instant disclosure.

The introduction of recombinant DNA constructs of the present disclosure into plants may be carried out by any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector-mediated DNA transfer, bombardment, or Agrobacterium-mediated transformation. Techniques for plant transformation and regeneration have been described in International Patent Publication WO 2009/006276, the contents of which are herein incorporated by reference.

In addition, methods to modify or alter the host endogenous genomic DNA are available. This includes altering the host native DNA sequence or a pre-existing transgenic sequence including regulatory elements, coding and non-coding sequences. These methods are also useful in targeting nucleic acids to pre-engineered target recognition sequences in the genome. As an example, the genetically modified cell or plant described herein, is generated using “custom” engineered endonucleases such as meganucleases produced to modify plant genomes (e.g., WO 2009/114321; Gao et al. (2010) Plant Journal 1:176-187). Another site-directed engineering is through the use of zinc finger domain recognition coupled with the restriction properties of restriction enzyme (e.g., Urnov, et al. (2010) Nat Rev Genet. 11(9):636-46; Shukla, et al. (2009) Nature 459 (7245):437-41). A transcription activator-like (TAL) effector-DNA modifying enzyme (TALE or TALEN) is also used to engineer changes in plant genome. See e.g., US20110145940, Cermak et al., (2011) Nucleic Acids Res. 39(12) and Boch et al., (2009), Science 326 (5959): 1509-12. Site-specific modification of plant genomes can also be performed using the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system. See e.g., Belhaj et al., (2013), Plant Methods 9: 39; The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA.

The development or regeneration of plants containing the foreign, exogenous isolated nucleic acid fragment that encodes a protein of interest is well known in the art. The regenerated plants may be self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant containing a desired polypeptide is cultivated using methods well known to one skilled in the art.

EXAMPLES

The present disclosure is further illustrated in the following examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these examples, while indicating embodiments of the disclosure, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Furthermore, various modifications of the disclosure in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1

Drought Tolerance Genes Cloning and Over-Expression Vector Construction

Based on our preliminary screening of rice activation tagging population and the sequence information of gene IDs shown in Table 2, primers were designed for cloning rice abiotic stress tolerance genes OsGSTU41, OsPPCK4, OsCAM2, OsDN-DTP4, OsLecRK4.1 and OsLecRK4.2. The primers and the expected-lengths of the amplified genes are shown in Table 3.

For OsGSTU41, OsPPCK4 and OsCAM2, their cDNAs were cloned by using pooled cDNA from leaf, stem and root tissues of Zhonghua 11 plant as the template. For OsDN-DTP4, OsLecRK4.1, and OsLecRK4.2, their gDNAs were cloned, and amplified using genomic DNA of Zhonghua 11 as the template. The PCR reaction mixtures and PCR procedures are shown in Table 4 and Table 5.

TABLE 2
Rice gene names, Gene IDs (from TIGR) and Construct IDs
	Gene name	LOC ID	Construct ID
	OsGSTU41	LOC_Os01g72160	DP0043
	OsPPCK4	LOC_Os02g56310	DP0058
	OsCAM2	LOC_Os05g41210	DP0059
	OsDN-DTP4	LOC_Os07g04720	DP0167
	OsLecRK4.1	LOC_Os04g44900	DP0173
	OsLecRK4.2	LOC_Os04g44910	DP0209

TABLE 3
Primers for cloning rice drought tolerance genes
				Length of
		SEQ		amplified
Primer	Sequence	ID NO:	Gene name	fragment (bp)
gc-371	5′-AAATGGTTAAGCTAATCAGCGCCTTC-3′	21	OsGSTU41	727
gc-372	5′-TACGCATATTCCAGCATCGAAATTCAC-3′	22
DEgc-516	5′-GGAGAAGAGAATCGCGGAGATAGC-3′	23	OsPPCK4	760
DEgc-517	5′-TGGAGTTCATCATCATCCTCGATC-3′	24
DEgc-546	5′-CTCGCGGAACCTTCTCGAAGCTTC-3′	25	OsCAM2	576
DEgc-547	5′-CAGCTTTGTTGTAGGCCCTGAC-3′	26
gc-996	5′-GCGGCAAAAACGATGTCAGTGGCTAG-3′	27	OsDN-DTP4	1184
gc-997	5′-GTCCCTTAACTATATAAAACCGGTCTCCC-3′	28
gc-1573	5′-ACCGGGGCCGTGACTTGACTGAC-3′	29	OsLecRK4.1	2346
gc-1574	5′-CGTCGACAATCAGATCAGAGGAGAA-3′	30
gc-1578	5′-GTAGCGAGGAGTGTGAACGATGTGATGC-3′	31	OsLecRK4.2	2464
gc-1579	5′-GCCTTCTCGAAGCTTTGCACACTCACTG-3′	32

TABLE 4
PCR reaction mixture for cloning drought tolerance genes
	Reaction mix	50 μL
	Template	1	μL
	TOYOBO KOD-FX (1.0 U/μL)	1	μL
	2 × PCR buffer for KOD-FX	25	μL
	2 mMdNTPs (0.4 mM each)	10	μL
	Primer-F/R (10 μM)	2	μL each
	ddH2O	9	μL

TABLE 5
PCR cycle conditions
	94° C.	3	min
	98° C.	10	s
	58° C.	30	s	{close oversize brace}	×30
	68° C.	(1 Kb/min) 1 min
	68° C.	5	min

The PCR amplified products were extracted after the agarose gel electrophoresis using a column kit and then ligated with TA cloning vectors. The sequences and orientation in these constructs were confirmed by sequencing. Then these genes were cloned into plant binary construct DP0005 (pCAMBIA1300-AsRed) (SEQ ID NO: 1) or DP0158 which was generated by transferring DsRed gene expression cassette (SEQ ID NO: 2 in the sequence list) into construct DP0005.

OsGSTU41, OsPPCK4 and OsCAM2 were cloned into the construct of DP0005. The generated over-expression vectors were listed in Table 2. The cloned nucleotide sequence in construct of DP0043 and coding sequence of OsGSTU41 are provided as SEQ ID NO: 3 and 4, the encoded amino acid sequence of OsGSTU41 is shown in SEQ ID NO: 5; the cloned nucleotide sequence in construct of DP0058 and coding sequence of OsPPCK4 are provided as SEQ ID NO: 6 and 7, the encoded amino acid sequence of OsPPCK4 is shown in SEQ ID NO: 8; the cloned nucleotide sequence in construct of DP0059 and coding sequence of OsCAM2 are provided as SEQ ID NO: 9 and 10, the encoded amino acid sequence of OsCAM2 is shown in SEQ ID NO: 11.

OsDN-DTP4, OsLecRK4.1 and OsLecRK4.2 were cloned into the construct of DP0158. The cloned nucleotide sequence in construct of DP0167 and coding sequence of OsDN-DTP4 are provided as SEQ ID NO: 12 and 13, the encoded amino acid sequence of OsDN-DTP4 is shown in SEQ ID NO: 14; the cloned nucleotide sequence in construct of DP0173 and coding sequence of OsLecRK4.1 are provided as SEQ ID NO: 15 and 16, the encoded amino acid sequence of OsLecRK4.1 is shown in SEQ ID NO: 17; and the cloned nucleotide sequence in construct of DP0209 and coding sequence of OsLecRK4.2 are provided as SEQ ID NO: 18 and 19, the encoded amino acid sequence of OsLecRK4.2 is shown in SEQ ID NO: 20.

Example 2

Generation of Transgenic Rice Lines

The over-expression vectors and empty vectors (DP0005 and DP0158) were transformed into the Zhonghua 11 (Oryza sativa L.) by Agrobacteria-mediated method as described by Lin and Zhang ((2005) Plant Cell Rep. 23:540-547). Zhonghua 11 was cultivated by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. The first batch of seeds used in this research was provided by Beijing Weiming Kaituo Agriculture Biotech Co., Ltd. Calli induced from embryos was transformed with Agrobacteria with the vector. The transgenic seedlings (T₀) generated in transformation laboratory are transplanted in the field to get T₁ seeds. The T₁ and T₂ seeds are stored at cold room (4° C.), and the T₂ seeds were used for following trait screening.

OsGSTU41, OsPPCK4 and OsCAM2 transgenic seeds did not show red color under green fluorescent light. The T₁ transgenic plants were selected by hygromycin by culturing the rice plants (from 1-2 cm in height) in 50 mg/L hygromycin solution, the survived plants (hygromycin-resistant) were planted in field to produce T₂ seeds. Only the hygromycin-resistant T₂ transgenic rice plants were used in trait screen.

OsDN-DTP4, OsLecRK4.1 and OsLecRK4.2 transgenic seeds which showed red color under green fluorescent light (transgenic seeds) were used in the following assays.

Example 3

Gene Expression Analysis

Transgene expression levels in the transgenic rice plants were analyzed. A standard RT-PCR or a real-time RT-PCR procedure, such as the QuantiTect® Reverse Transcription Kit from Qiagen® and Real Time-PCR (SYBR^RPremix Ex Taq™, TaKaRa), is used. EF-1α gene is used as an internal control to show that the amplification and loading of samples from the transgenic rice and wild-type are similar. Gene expression is normalized based on the EF-1α mRNA levels.

As shown in FIG. 3, the expression level of OsGSTU41 gene in DP0043.15 rice is set at 1.00, OsGSTU4/over-expressed in all the ten transgenic rice lines, while the expression of OsGSTU41 was not detected in ZH11-TC plants. The primers used for the real-time PCR are as below:

(SEQ ID NO: 33)
DP0043-3: 5′-GGCTGTCGTTCTGGATGG-3′

(SEQ ID NO: 34)
DP0043-4: 5′-GCAGTGAACAAGGCGACG-3′

As shown in FIG. 4, the expression level of OsPPCK4 gene in ZH11-TC rice is set at 1.00, OsPPCK4 over-expressed in all the transgenic lines, while the expression levels of OsPPCK4 were very low in both ZH11-TC and DP0158 controls.

(SEQ ID NO: 35)
DP0058-F1: 5′-GCTCTACATGATGCTCTCCG-3′

(SEQ ID NO: 36)
DP0058-R1: 5′-GAGACGTCCTTGCAGAGC-3′

The expression level of OsDN-DTP4 gene in ZH11-TC rice is set at 1.00, OsDN-DTP4 over-expressed in all the nine transgenic rice lines (FIG. 5).

(SEQ ID NO: 37)
DP0167-F1: 5′-CCAGTTCAGAGTACGGTGCCG-3′

(SEQ ID NO: 38)
DP0167-R1: 5′-GTGTCCACGTCAGCCTCCTTTC-3′

The expression level of OsLecRK4.1 gene in ZH11-TC rice is set at 1.00, OsLecRK4.1 over-expressed in all the nine transgenic rice lines (FIG. 6).

(SEQ ID NO: 39)
DP0173-F1: 5′-CGCTCAACATCTCATCCC-3′

(SEQ ID NO: 40)
DP0173-R1: 5′-CCGCATGAACACGAACAC-3′

As shown in FIG. 7, the expression level of OsLecRK4.2 gene in ZH11-TC rice is set at 1.00, OsGLRL1.2 over-expressed in all the transgenic lines, while the expression levels of OsLecRK4.2 were very low in ZH11-TC plants.

(SEQ ID NO: 41)
DP0209-F1: 5′-CCGACGATGGTGAGCTAC-3′

(SEQ ID NO: 42)
DP0209-R1: 5′-GTGACGGTGGAAGGGAAG-3′

Example 4

Drought Screening of Transgenic Rice Plants

The transgenic rice plants were screened in greenhouse drought assays. Two types of lamps, i.e. sodium lamp and metal halide lamp with the ratio of 1:1, were provided as light source with a 16-h-light/8-h-dark cycle and placed approximately 1.5 m height above the seedbed. The light intensity 30 cm above the seedbed was measured as 10,000-20,000 lx in sunny day, while 6,000-10,000 lx in cloudy day, the relative humidity ranged from 30% to 90%, and the temperature ranged from 20 to 35° C.

Drought Screening Method:

T₂ Transgenic seeds were sterilized by 800 ppm carbendazol for 8 h at 32° C. and washed 3-5 times with distilled water, then soaked in water for 16 h at 32° C., germinated for 18 h at 35-37° C. in an incubator. The germinated seeds were sowed in one tray or pot filled with mixture of organic soil, vermiculite and sand (V:V:V=3:3:2). The seedlings were grown under normal greenhouse condition and watered by modified IRRI solution. When the seedlings grew to 3-leaf stage, watering was stopped and the trays were kept into a dry place until the leaves became dry and curved (approximately 9-15 days depending on the seasons). The trays were transferred into water pool to recover the seedlings for 5-7 days, and then the plants were scored for the recovery degree. The following scoring system was used: more than half green stem=1, more than two third green leaf=1, less than two third but more than one third green leaf=0.5, less than one third green leaf=0.2, no green leaf or less than half green stem=0. The recovery degree was the sum of the score of the green tissues, and the data were statistically analyzed using Mixed Model. The lines showed significant better than the controls (P<0.05) were considered as positive ones. Survival rate (percentage of survived plants over the total plant number) was also used as a parameter for drought screening.

Two experimental designs were used. (1) Latin Square Design was used, and the total 16 plants for each line grew into different positions of the tray. The wild-type control (Zhonghua 11) from tissue culture procedure (ZH11-TC) and/or empty vector (DP0158) transgenic control in the same tray were used as controls. Several positive control (a drought tolerant variety, Mianhui 501) and negative control (a drought sensitive variety, Dongbeiyin 2) seedlings also were planted in the same tray. (2) Randomized Block Design was used for confirming the observation of the transformed rice from construct level. Nine to twelve transgenic lines from the same construct were planted in one experimental unit to evaluate the transgene at construct level by Mixed Model considering construct, line and environment effects. If the survival rates or recovery degrees of the transgenic rice plants were significantly greater than that of control (P<0.05), the gene was considered as having drought tolerant function.

GH Drought Assay Results:

1) GH DRT Validation Results of OsGSTU41 (DP0043) Transgenic Rice

Ten OsGSTU41 transgenic lines were tested by Latin square design in the first experiment. Different lines were planted in different trays, and the ZH11-TC and DP0005 seedlings in the same tray were used as their corresponding controls. Table 6 shows that all of the ten lines exhibited higher survival rates and recovery degrees, and six lines showed significantly higher recovery degrees than ZH11-TC control. When compared with the DP0005 control, eight lines exhibited higher survival rates and average recovery degrees, and four lines showed significantly higher recovery degrees. These results demonstrate that OsGSTU41 transgenic rice had enhanced drought tolerance at seedling stage.

TABLE 6
Enhanced drought tolerance of OsGSTU41transgenic rice
plants under greenhouse conditions (1st experiment)
	Number of	Number		Average
	survived	of total	Survival	recovery		P ≤
Line ID	plants	plants	rate (%)	degree	Pvalue	0.05
DP0043.02	6	16	37.5	0.45	0.1373
ZH11-TC	3	16	18.8	0.19
DP0043.03	9	16	56.3	0.63	0.2394
ZH11-TC	5	16	31.3	0.38
DP0043.11	7	16	43.8	0.61	0.5978
ZH11-TC	5	16	31.3	0.47
DP0043.12	11	16	68.8	0.70	0.0004	Y
ZH11-TC	1	16	6.3	0.09
DP0043.15	12	16	75.0	1.06	0.0007	Y
ZH11-TC	5	15	33.3	0.31
DP0043.19	10	16	62.5	0.66	0.4345
ZH11-TC	5	12	41.7	0.50
DP0043.25	12	16	75.0	1.38	0.0022	Y
ZH11-TC	5	14	35.7	0.47
DP0043.26	14	16	87.5	0.99	0.0004	Y
ZH11-TC	2	13	15.4	0.16
DP0043.28	14	16	87.5	1.19	0.0017	Y
ZH11-TC	4	16	25.0	0.37
DP0043.29	13	16	81.3	1.59	0.0010	Y
ZH11-TC	8	15	53.3	0.76

In the second experiment, construct level design was used, and nine transgenic lines were tested. When grown to 3-leaf stage, the plants were first drought stressed for 17 days, recovered in water for six days, and then were drought stressed for 22 days and recovered for seven days. 69 of the 108 OsGSTU41 transgenic rice plants survived, while 9 of the 24 ZH11-TC and 5 of the 12 DP0158 seedlings survived. OsGSTU41 transgenic rice exhibited higher survival rate and average recovery degree than both ZH11-TC and DP0158 seedlings at the construct level (Table 7). Analysis at transgenic line level showed that eight lines exhibited higher survival rates and average recovery degrees than both controls (Table 8). These results indicate that OsGSTU41 transgenic rice showed enhanced drought tolerance at seedling stage, and OsGSTU41 plays a role in improving drought tolerance of transgenic plants.

TABLE 7
Enhanced drought tolerance of OsGSTU41transgenic rice plants under
greenhouse conditions at construct level (2nd experiment)
	Number of	Number		Average
	survived	of total	Survival	recovery		P ≤
Construct ID	plants	plants	rate (%)	degree	P value	0.05
DP0043	69	108	63.9	0.64	0.0426	Y
ZH11-TC	9	24	37.5	0.40
DP0043	69	108	63.9	0.64	0.1615
DP0158	5	12	41.7	0.42

TABLE 8
Enhanced drought tolerance of OsGSTU41transgenic rice plants
under greenhouse conditions at line level (2nd experiment)
	Number of	Number		Average
	survived	of total	Survival	recovery	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	rate (%)	degree	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0043.06	3	12	25.0	0.52	0.3682		0.5479
DP0043.11	9	12	75.0	0.69	0.0381	Y	0.1238
DP0043.12	6	12	50.0	0.60	0.1493		0.3011
DP0043.13	9	12	75.0	0.68	0.0474	Y	0.1425
DP0043.15	7	12	58.3	0.63	0.1047		0.2387
DP0043.19	8	12	66.7	0.65	0.0714		0.1860
DP0043.24	8	12	66.7	0.65	0.0714		0.1860
DP0043.26	9	12	75.0	0.68	0.0474	Y	0.1425
DP0043.28	10	12	83.3	0.70	0.0304	Y	0.1071
ZH11-TC	9	24	37.5	0.40
DP0158	5	12	41.7	0.42

2) GH DRT Validation Results of OsPPCK4 (DP0058) Transgenic Rice

Twelve OsPPCK4 transgenic lines were tested by drought stress, and planted in different trays in the first experiment. ZH11-TC plants in the same tray were used as their corresponding controls. Table 9 shows that ten lines exhibited higher survival rates and recovery degrees than that of ZH11-TC control, and five lines showed significantly higher recovery degrees, indicating that the OsPPCK4 transgenic rice plants had improved drought tolerance at seedling stage.

TABLE 9
Enhanced drought tolerance of OsPPCK4transgenic rice
plants under greenhouse conditions (1st experiment)
	Number of	Number		Average
	survived	of total	Survival	recovery		P ≤
Line ID	plants	plants	rate (%)	degree	Pvalue	0.05
DP0058.02	8	16	50.0	0.54	0.0119	Y
ZH11-TC	1	16	6.3	0.06
DP0058.03	5	16	31.3	0.31	0.1701
ZH11-TC	2	16	12.5	0.13
DP0058.04	7	16	43.8	0.56	0.0219	Y
ZH11-TC	2	16	12.5	0.13
DP0058.07	11	16	68.8	0.81	0.5165
ZH11-TC	9	16	56.3	0.66
DP0058.08	7	16	43.8	0.44	0.8143
ZH11-TC	5	16	31.3	0.38
DP0058.10	5	16	31.3	0.50	0.6151
ZH11-TC	8	16	50.0	0.64
DP0058.11	11	15	73.3	2.16	0.2430
ZH11-TC	9	15	60.0	1.56
DP0058.13	10	16	62.5	1.43	0.0020	Y
ZH11-TC	0	16	0.0	0.00
DP0058.14	9	16	56.3	1.45	0.7110
ZH11-TC	6	16	37.5	1.26
DP0058.15	9	15	60.0	1.09	0.0131	Y
ZH11-TC	3	16	18.8	0.28
DP0058.18	12	16	75.0	2.33	0.0045	Y
ZH11-TC	5	16	31.3	0.96

In the second experiment, construct level design was used, and nine transgenic lines were tested. When grown to 3-leaf stage, the plants were drought stressed for 17 days, recovered in water for seven days. 49 of the 108 OsPPCK4 transgenic rice plants survived, whereas 6 of the 24 ZH11-TC and 1 of the 12 DP0158 seedlings survived. OsPPCK4 transgenic rice exhibited higher survival rate and exhibited significantly higher average recovery degree than both ZH11-TC and DP0158 seedlings at the construct level (Table 10). Analysis at line level showed that six lines exhibited higher survival rates and nine lines exhibited higher recovery degrees than both controls (Table 11). These results demonstrate that OsPPCK4 transgenic rice showed enhanced drought tolerance at seedling stage and OsPPCK4 plays a role in improving drought tolerance of transgenic plants.

TABLE 10
Enhanced drought tolerance of OsPPCK4 transgenic rice plants under
greenhouse conditions at construct level (2nd experiment)
	Number of	Number	Survival	Average
Construct	survived	of	rate	recovery
ID	plants	total plants	(%)	degree	P value	P ≤ 0.05
DP0058	49	108	45.4	0.66	0.0479	Y
ZH11-TC	6	24	25.0	0.32
DP0058	49	108	45.4	0.66	0.0102	Y
DP0158	1	12	8.3	0.08

TABLE 11
Enhanced drought tolerance of OsPPCK4 transgenic rice plants under
greenhouse conditionsat transgenic line level (2nd experiment)
	Number of	Number		Average
	survived	of total	Survival	recovery	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	rate (%)	degree	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0058.01	8	12	66.7	0.77	0.0269	Y	0.0059	Y
DP0058.03	3	12	25.0	0.51	0.3440		0.0846
DP0058.04	7	12	58.3	0.73	0.0405	Y	0.0088	Y
DP0058.05	5	12	41.7	0.69	0.0665		0.0144	Y
DP0058.06	6	12	50.0	0.63	0.1200		0.0265	Y
DP0058.10	3	12	25.0	0.53	0.3037		0.0732
DP0058.13	8	12	66.7	0.77	0.0247	Y	0.0054	Y
DP0058.15	3	12	25.0	0.54	0.2668		0.0631
DP0058.16	6	12	50.0	0.73	0.0438	Y	0.0095	Y
ZH11-TC	6	24	25.0	0.32			0.3444
DP0158	1	12	8.3	0.08

3) GH DRT Validation Results of OsCAM2 (DP0059) Transgenic Rice

In the first experiment, Latin square design was used, and eight OsCAM2 transgenic lines were tested. Different lines were planted in different trays, and the ZH11-TC seedlings in the same tray were used as their corresponding controls. Table 12 shows that six lines had higher survival rates and recovery degrees than that of ZH11-TC control, and four of which showed significantly higher recovery degrees, indicating that the OsCAM2 transgenic rice plants had improved drought tolerance at seedling stage.

In the second experiment, construct level testing design was used, and the eight OsCAM2 transgenic lines were tested again. As shown in Table 13, 83 of the 96 OsCAM2 transgenic seedlings survived after drought stress, and the survival rate and recovery degree was higher than that of DP0158 control (P value=0.0679) and significantly higher than that of ZH11-TC control (P value=0.0090). Analysis at transgenic line level showed that all the eight lines exhibited higher survival rates and average recovery degrees, five lines showed significantly higher recovery degrees than ZH11-TC control and three lines showed significantly higher recovery degrees than DP0158 control (Table 14). These results further demonstrate that OsCAM2 gene enhances drought tolerance in plant.

TABLE 12
Enhanced drought tolerance of OsCAM2transgenic rice plants
under greenhouse conditions (1st experiment)
	Number	Number
	of	of	Survival	Average
	survived	total	rate	recovery
Line ID	plants	plants	(%)	degree	Pvalue	P ≤ 0.05
DP0059.01	8	16	50.0	0.58	0.0100	Y
ZH11-TC	0	15	0.0	0.00
DP0059.04	10	16	62.5	1.03	0.5592
ZH11-TC	6	15	40.0	0.89
DP0059.05	11	16	68.8	0.93	0.1365
ZH11-TC	4	16	25.0	0.50
DP0059.09	11	16	68.8	1.23	0.0239	Y
ZH11-TC	9	16	56.3	0.60
DP0059.11	14	16	87.5	1.28	0.0252	Y
ZH11-TC	9	15	60.0	0.76
DP0059.12	3	16	18.8	0.76	0.4943
ZH11-TC	5	16	31.3	1.17
DP0059.13	4	16	25.0	0.28	0.2387
ZH11-TC	9	16	56.3	0.60
DP0059.14	14	15	93.3	1.64	0.0003	Y
ZH11-TC	9	15	60.0	0.76

TABLE 13
Enhanced drought tolerance of OsCAM2transgenic rice plants
under greenhouse conditions at construct level (2nd experiment)
	Number of	Number	Survival	Average
Construct	survived	of total	rate	recovery
ID	plants	plants	(%)	degree	Pvalue	P ≤ 0.05
DP0059	83	96	86.5	1.05	0.0090	Y
ZH11-TC	15	23	65.2	0.68
DP0059	83	96	86.5	1.05	0.0679
DP0158	7	12	58.3	0.71

TABLE 14
Enhanced drought tolerance of OsCAM2transgenic rice plants under greenhouse
conditions at line level (2nd experiment)
	Number of	Number		Average
	survived	of total	Survival	recovery	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	rate (%)	decree	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0059.01	11	12	91.7	1.00	0.0535		0.1576
DP0059.04	9	12	75.0	0.94	0.1143		0.2599
DP0059.05	12	12	100.0	1.13	0.0066	Y	0.0399	Y
DP0059.09	12	12	100.0	1.18	0.0030	Y	0.0240	Y
DP0059.11	10	12	83.3	1.02	0.0419	Y	0.1341
DP0059.12	8	12	66.7	0.92	0.1401		0.2975
DP0059.13	10	12	83.3	1.07	0.0190	Y	0.0800
DP0059.14	11	12	91.7	1.13	0.0070	Y	0.0415	Y
ZH11-TC	15	23	65.2	0.68
DP0158	7	12	58.3	0.71

In the third experiment, construct level design was also used, and eight transgenic lines were tested. When grown to 3-leaf stage, the plants were drought stressed for 15 days, recovered in water for seven days. 55 of the 96 OsCAM2 transgenic rice plants survived, while 4 of the 24 ZH11-TC and 4 of the 12 DP0158 seedlings survived. OsCAM2 transgenic rice exhibited higher survival rate and exhibited significantly higher average recovery degree than both ZH11-TC seedlings at the construct level (Table 15). Analysis at line level showed that all the eight lines exhibited higher survival rates and average recovery degrees than both controls (Table 16). In these three experiments, OsCAM2 transgenic rice showed enhanced drought tolerance at seedling stage. These results demonstrate that OsCAM2 plays a role in improving drought tolerance of transgenic plants.

TABLE 15
Enhanced drought tolerance of OsCAM2transgenic rice plants
under greenhouse conditions at the construct level (3rd experiment)
	Number of	Number	Survival	Average
Construct	survived	of	rate	recovery
ID	plants	total plants	(%)	degree	P value	P ≤ 0.05
DP0059	55	96	57.3	0.59	0.0004	Y
ZH11-TC	4	24	16.7	0.17
DP0059	55	96	57.3	0.59	0.1012
DP0158	4	12	33.3	0.33

TABLE 16
Enhanced drought tolerance of OsCAM2transgenic rice plants under greenhouse
conditions at line level (3rd experiment)
	Number
	of	Number		Average
	survived	of total	Survival	recovery	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	rate (%)	degree	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0059.01	10	12	83.3	0.67	0.0002	Y	0.0497	Y
DP0059.04	7	12	58.3	0.59	0.0020	Y	0.1346
DP0059.05	8	12	66.7	0.61	0.0010	Y	0.0985
DP0059.09	6	12	50.0	0.56	0.0038	Y	0.1801
DP0059.11	9	12	75.0	0.65	0.0003	Y	0.0594
DP0059.12	5	12	41.7	0.53	0.0069	Y	0.2359
DP0059.13	4	12	33.3	0.51	0.0121	Y	0.3032
DP0059.14	6	12	50.0	0.59	0.0020	Y	0.1346
ZH11-TC	4	24	16.7	0.17
DP0158	4	12	33.3	0.33

4) GH DRT Validation Results of OsDN-DTP4 (DP0167) Transgenic Rice

In the first experiment, Latin square design was used, and 12 OsDN-DTP4 transgenic lines were tested. Different lines were planted in different trays, and the ZH11-TC seedlings in the same tray were used as their corresponding controls. Table 17 shows that eight lines had higher survival rates and recovery degrees, and four lines had significantly higher recovery degrees than that of ZH11-TC control. These results indicate that the OsDN-DTP4 transgenic rice plants had improved drought tolerance at seedling stage.

Nine lines were tested in the second and third experiments using construct level design. In the second experiment, as shown in Table 18, all the tested OsDN-DTP4 transgenic rice plants exhibited higher survival rate and recovery degree than that of DP0158 and ZH11-TC controls at the construct level. And further analysis at line level indicated that all of the 9 lines showed higher survival rates and recovery degrees than that of either ZH11-TC or DP0158 control (Table 19). And the experimental results in the third experiment, showed the same tendency (Table 20 and 21). The recovery degree of all the tested OsDN-DTP4 transgenic rice plants had higher survival rate and recovery degree than that of ZH11-TC and DP0158 controls both at construct level and at transgenic line level. All these results further demonstrate that OsDN-DTP4 gene enhances drought tolerance in plant.

TABLE 17
Drought tolerance assayof OsDN-DTP4transgenic rice plants under greenhouse
conditions (1st experiment)
	Number of	Number	Survival	Average
	survived	of total	rate	recovery
Line ID	plants	plants	(%)	degree	Pvalue	P ≤ 0 .05
DP0167.01	11	16	68.8	0.93	0.8279
ZH11-TC	10	16	62.5	0.89
DP0167.02	9	16	56.3	0.81	0.0208	Y
ZH11-TC	2	16	12.5	0.13
DP0167.03	9	16	56.3	1.12	0.0227	Y
ZH11-TC	2	16	12.5	0.25
DP0167.04	3	16	18.8	0.34	0.5673
ZH11-TC	2	16	12.5	0.19
DP0167.05	8	16	50.0	0.73	0.0186	Y
ZH11-TC	1	15	6.7	0.13
DP0167.06	9	16	56.3	1.03	0.1491
ZH11-TC	4	15	26.7	0.56
DP0167.07	6	16	37.5	0.45	0.7690
ZH11-TC	6	15	40.0	0.38
DP0167.08	13	16	81.3	1.64	0.1871
ZH11-TC	12	16	75.0	1.22
DP0167.09	11	16	68.8	0.69	0.0038	Y
ZH11-TC	4	16	25.0	0.25
DP0167.11	8	16	50.0	0.61	0.8116
ZH11-TC	6	16	37.5	0.67
DP0167.13	8	16	50.0	0.51	0.7480
ZH11-TC	9	16	56.3	0.56

TABLE 18
Drought tolerance assayof OsDN-DTP4transgenic rice plants
under greenhouse conditions at construct level (2nd experiment)
	Number of	Number	Survival	Average
Construct	survived	of total	rate	recovery
ID	plants	plants	(%)	degree	Pvalue	P ≤ 0.05
DP0167	84	108	77.8	0.97	0.1807
ZH11-TC	16	24	66.7	0.75
DP0167	84	108	77.8	0.97	0.0823
DP0158	5	12	41.7	0.58

TABLE 19
Drought tolerance assayof OsDN-DTP4transgenic rice plants under greenhouse
conditions at line level (2nd experiment)
	Number of	Number		Average
	survived	of total	Survival	recovery	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	rate (%)	degree	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0167.01	11	12	91.7	0.97	0.1807		0.0823
DP0167.02	8	12	66.7	0.97	0.1807		0.0823
DP0167.03	11	12	91.7	0.97	0.1807		0.0823
DP0167.04	8	12	66.7	0.97	0.1807		0.0823
DP0167.05	9	12	75.0	0.97	0.1807		0.0823
DP0167.06	10	12	83.3	0.97	0.1807		0.0823
DP0167.07	9	12	75.0	0.97	0.1807		0.0823
DP0167.11	9	12	75.0	0.97	0.1807		0.0823
DP0167.12	9	12	75.0	0.97	0.1807		0.0823
ZH11-TC	16	24	66.7	0.75
DP0158	5	12	41.7	0.58

TABLE 20
Drought tolerance assayof OsDN-DTP4transgenic rice plants
under greenhouse conditions at construct level (3rd experiment)
	Number of	Number of		Average
Construct	survived	total	Survival	recovery
ID	plants	plants	rate (%)	degree	P value	P ≤ 0.1
DP0167	55	108	50.9	0.69	0.6312
ZH11-TC	11	24	45.8	0.61
DP0167	55	108	50.9	0.69	0.0561	Y
DP0158	3	12	25.0	0.27

TABLE 21
Drought tolerance assayof OsDN-DTP4transgenic rice plants under greenhouse
conditions at line level (3rd experiment)
	Number of	Number		Average
	survived	of total	Survival	recovery	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	rate (%)	degree	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0167.01	7	12	58.3	0.69	0.6312		0.0561
DP0167.02	6	12	50.0	0.69	0.6312		0.0561
DP0167.03	6	12	50.0	0.69	0.6312		0.0561
DP0167.04	8	12	66.7	0.69	0.6312		0.0561
DP0167.05	6	12	50.0	0.69	0.6312		0.0561
DP0167.06	4	12	33.3	0.69	0.6312		0.0561
DP0167.07	5	12	41.7	0.69	0.6312		0.0561
DP0167.11	6	12	50.0	0.69	0.6312		0.0561
DP0167.12	7	12	58.3	0.69	0.6312		0.0561
ZH11-TC	11	24	45.8	0.61
DP0158	3	12	25.0	0.27

Example 5

Field Drought Tolerance Assays of Mature Transgenic Rice Plants

Drought stress at flowering stage is an important problem in agriculture practice. The transgenic rice plants were further tested under field drought conditions. For the field drought assays of mature rice plants, 9-12 transgenic lines of each construct were tested. The T₂ seeds were first sterilized as described in Example 4. The germinated seeds were planted into a seedbed. The seedlings were transplanted into the testing field at 3-leaf stage with 3-4 replicates. About 8-10 plants per replicate for each transgenic line were planted into the same block. And the ZH11-TC, DP0158, Bulk Null or Line Null planted nearby the transgenic lines of the same block, were used as the controls in statistical analysis according to the experimental design.

The rice plants were managed by normal practice using pesticides and fertilizers. Watering was stopped at the panicle initiation stage II stage, so as to give drought stress at flowering stage depending on the weather conditions (temperature and humidity). The soil water content was measured every four days at several sites of each block using TDR30 (Spectrum Technologies, Inc.).

Plant phenotypes, included heading date, leaf rolling degree, drought sensitivity and drought tolerance, were observed and recorded during the experimental processes. Special attention was paid to the leaf rolling degree at noontime. At the end of the planting season, six representative plants of each transgenic line were harvested from the middle of the row per line, and the grain weight per plant was measured. The grain weight data were statistically analyzed using mixed linear model. Positive transgenic lines were selected based on the analysis.

Field Drought Assay Results:

1) Field DRT Validation Results of OsGSTU41 (DP0043) Transgenic Rice

Twenty-two OsGSTU41 transgenic lines were tested in Hainan Province in the first field experiment using the corresponding line null planted every two rows as the control. Four replicates per transgenic line and 10 plants per replicate were planted into the same block. Watering was stopped from panicle initiation stage II to seed mature to produce heavier drought stress. The soil volumetric moisture content decreased from about 30% to 10% during the heading and maturation stages (FIG. 1). At the end of the planting season, six representative plants of each transgenic line were harvested from the middle of the row per line, and the grain weight per plant was measured. Thirteen lines exhibited higher grain yield per plant than that of their corresponding line null control, and seven lines exhibited significantly higher at the level of P<0.05 as shown in Table 22. These results demonstrate that OsGSTU41 transgenic rice plants had greater grain yield per plant than that of the control after drought stress.

Twelve OsGSTU41 transgenic lines were tested again in Hainan Province in the second field experiment. The bulk null planted nearby were used as the control. And three replicates per transgenic line and eight plants per replicate were planted into the same block. Watering was stopped from panicle initiation stage II to seed mature to produce heavier drought stress. The soil volumetric moisture content decreased from about 27% to 6% during the heading and maturation stages (FIG. 2). Three transgenic lines DP0043.10, DP0043.16 and DP0043.22 showed drought tolerance phenotype such as less leaf roll degree and greener leaf. Four lines DP0043.10, DP0043.16, DP0043.22 and DP0043.26 showed better seed setting rates at the maturation stage. At the end of the planting season, six representative plants of each transgenic line were harvested from the middle of the row per line, and the grain weight per plant was measured. As shown in Table 23, 11 OsGSTU41 transgenic lines exhibited higher grain yield per plant than that of the bulk null control, and two lines showed significantly higher at the level of P<0.1. These results further demonstrate that OsGSTU41 transgenic rice plants had greater grain yield per plant than that of the control after drought stress.

As described in Example 4, OsGSTU41 transgenic rice exhibited enhanced drought tolerance at seedling stage. These results at mature stage further demonstrate OsGSTU41 gene plays a role in enhancing drought tolerance in plant from seedling stage to mature stage.

TABLE 22
Grain yield analysisof OsGSTU41transgenic rice plants
under field drought conditions(1st experiment)
	Number	Number	Grain
	of	of	yield
	survived	harvest	per plant
Line ID	plants	plants	(g)	Pvalue	P ≤ 0.05
DP0043.06	40	24	4.93	0.993
DP0043.06-Null	40	24	4.59
DP0043.10	40	24	4.36	0.008	Y
DP0043.11-Null	40	24	2.42
DP0043.11	40	24	2.75	0.491
DP0043.11-Null	40	24	2.42
DP0043.12	40	24	3.49	0.455
DP0043.12-Null	40	24	3.00
DP0043.13	40	24	5.63	0.022	Y
DP0043.13-Null	40	24	2.91
DP0043.16	40	24	3.57	0.000	Y
DP0043.16-Null	40	24	0.11
DP0043.17	40	24	6.88	0.000	Y
DP0043.17-Null	40	24	1.99
DP0043.21	40	24	5.32	0.013	Y
DP0043.21-Null	40	24	2.94
DP0043.22	40	24	4.02	0.048	Y
DP0043.22-Null	40	24	1.92
DP0043.23	40	24	6.65	0.457
DP0043.23-Null	40	24	5.98
DP0043.24	40	24	3.89	0.884
DP0043.24-Null	40	24	3.61
DP0043.26	40	24	7.77	0.026	Y
DP0043.26-Null	40	24	6.01
DP0043.29	40	24	4.33	0.098
DP0043.29-Null	40	24	2.56

TABLE 23
Grain yield analysisof OsGSTU41transgenic rice plants
under field drought conditions(2ndexperiment)
	Number	Number	Grain
	of	of	yield
	survived	harvest	per plant	CK = Bulk Null
Line ID	plants	plants	(g)	Pvalue	P ≤ 0.1
DP0043.02	24	15	5.19	0.464
DP0043.03	24	16	4.69	0.766
DP0043.04	24	14	4.73	0.747
DP0043.06	24	15	4.86	0.647
DP0043.10	24	15	4.74	0.731
DP0043.13	24	16	4.97	0.588
DP0043.16	24	16	6.43	0.061	Y
DP0043.17	24	15	4.87	0.658
DP0043.21	24	16	4.82	0.673
DP0043.22	24	16	4.78	0.720
DP0043.26	24	15	6.77	0.031	Y
DP0043.29	24	16	4.36	0.988
CK (Bulk Null)	24	16	4.38

2) Field DRT Validation Results of OsPPCK4 (DP0058) Transgenic Rice

Nine OsPPCK4 transgenic lines were tested in Beijing in the first field experiment using ZH11-TC and DP0158 rice plants as the controls. Three replicates per transgenic line and eight plants per replicate were planted into the same block. Watering was stopped from panicle initiation stage II to seed mature to produce heavier drought stress. The soil volumetric water content decreased from about 50% to 10% during the heading and maturation stages (FIG. 8). The transgenic line DP0058.14 showed drought tolerance phenotype such as keeping leaf expanding and leaf green. At the end of the planting season, about six representative plants of each transgenic line were harvested from the middle of the row per line, and the grain weight per plant was measured. Nine lines exhibited higher grain yield per plant than DP0158 control, and four lines exhibited higher grain yield per plant than ZH11-TC plants as shown in Table 24. These results demonstrate that OsPPCK4 transgenic rice plants had greater grain yield per plant than that of the control after drought stress.

TABLE 24
Grain yield analysis of OsPPCK4 transgenic rice plants under field drought
conditions
	Number of	Number of	Grain yield
	survived	harvested	per plant	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	(g)	P value	P ≤ 0.1	P value	P ≤ 0.1
DP0058.02	24	14	6.90	0.044		0.945
DP0058.07	24	16	7.88	0.220		0.407
DP0058.08	24	18	9.58	0.928		0.029	Y
DP0058.10	24	17	9.94	0.709		0.014	Y
DP0058.12	24	17	9.56	0.943		0.032	Y
DP0058.13	24	17	9.14	0.807		0.079	Y
DP0058.14	23	16	10.04	0.663		0.013	Y
DP0058.15	24	17	7.08	0.069		0.839
DP0058.18	24	17	7.82	0.211		0.442
ZH11-TC	24	18	9.47
DP0158	24	17	6.81
DP0043			8.66	0.45		0.08	Y
(construct)

3) Field DRT Validation Results of OsCAM2 (DP0059) Transgenic Rice

Eight OsCAM2 transgenic lines were tested in Beijing in the first field experiment using ZH11-TC and DP0158 rice plants as the controls. Three replicates per transgenic line and eight plants per replicate were planted into the same block. Watering was stopped from panicle initiation stage II to seed mature to produce heavier drought stress. The soil volumetric moisture content decreased from about 50% to 10% during the heading and maturation stages (FIG. 9). DP0059.05 showed better drought tolerance phenotype and better seed setting rate than control during drought stress. At the end of the planting season, about six representative plants of each transgenic line were harvested from the middle of the row per line, and the grain weight per plant was measured. The OsCAM2 transgenic rice exhibited significantly greater grain yield per plant than DP0158 control at the construct level. Eight lines exhibited higher grain yield per plant than DP0158 control, and three lines exhibited higher grain yield per plant than ZH11-TC plants as shown in Table 25. These results demonstrate that OsCAM2 transgenic rice plants had greater grain yield per plant than that of the control after drought stress.

TABLE 25
Grain yield analysis of OsCAM2 transgenic rice plants under field drought
conditions(1stexperiment)
	Number of	Number of	Grain yield
	survived	harvest	per	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	plant (g)	P value	P ≤ 0.1	P value	P ≤ 0.1
DP0059.01	24	17	9.42	0.969		0.048	Y
DP0059.04.	24	17	8.77	0.592		0.135
DP0059.05	24	18	10.09	0.624		0.011	Y
DP0059.09	24	17	9.16	0.811		0.066	Y
DP0059.11	24	18	10.12	0.617		0.011	Y
DP0059.12	24	18	8.81	0.611		0.123
DP0059.13	24	15	8.82	0.622		0.125
DP0059.14	24	12	10.57	0.404		0.005	Y
ZH11-TC	24	17	9.47
DP0158	24	16	6.81
DP0059			9.47	1.00		0.015	Y
(construct)

The second experiment was performed in Hainan province; the same eight OsCAM2 transgenic lines were tested. ZH11-TC and DP0158 rice plants were used as control. Ten plants from each line were planted and repeated for four times. Watering was stopped from panicle initiation stage II to seed maturity to produce heavier drought stress. The soil volumetric water content decreased from 35% to 5% during heading and maturation stage (FIG. 10). Five lines DP0059.04, DP0059.05, DP0059.09, DP0059.11 and DP0059.14 showed drought tolerance phenotype, the leaf rolling degree was less than control and the leaves were greener. At the end of the growing season, OsCAM2 transgenic rice did not show greater grain yield per plant, only two lines showed greater grain yields per plant (Table 26). These results demonstrate that OsCAM2 over-expressed transgenic rice plant obtained drought tolerance at seedling stage and over-expression of OsCAM2 improved the drought tolerance.

TABLE 26
Grain yield analysis of OsCAM2 transgenic rice plants under
field drought conditions(2nd experiment)
	Number	Number	Yield	CK =	CK =
	of	of	per	ZH11-TC	DP0158
	survival	harvested	plant	P	P ≤	P	P ≤
Line ID	plants	plants	(g)	value	0.1	value	0.1
DP0059.01	40	24	4.48	0.017		0.024
DP0059.04	40	24	5.68	0.449		0.565
DP0059.05	40	24	5.06	0.109		0.168
DP0059.09	40	24	6.27	0.994		0.862
DP0059.11	40	24	5.55	0.367		0.471
DP0059.12	39	24	5.25	0.177		0.259
DP0059.13	40	24	4.99	0.089		0.127
DP0059.14	39	24	6.79	0.514		0.400
ZH11-TC	40	24	6.27
DP0158	39	24	6.13
DP0059			5.51	0.313		0.413
(construct)

4) Field DRT Validation Results of OsLecRK4.1 (DP0173) Transgenic Rice

Twelve OsLecRK4.1 transgenic lines were tested in the first field experiment using ZH11-TC and DP0158 rice plants as the controls. Four replicates per transgenic line and ten plants per replicate were planted into the same block. Watering was stopped from panicle initiation stage II to seed mature to produce heavier drought stress. The soil volumetric moisture content decreased from about 40% to 5% during the heading and maturation stages (FIG. 11). Four transgenic lines DP0173.01, DP0173.04, DP0173.08 and DP0173.14 showed greener leaf and less leaf roll degree compared with the ZH11-TC and DP0158 controls planted nearby. DP0173.04 also showed better seed setting rate at the maturation stage. The grain weight per plant of OsLecRK4.1 transgenic rice was less than ZH11-TC but more than DP0158 control at the construct level. Transgenic line level analysis is shown in Table 27. These results demonstrate that OsLecRK4.1 transgenic rice plants had improved drought tolerance at seedling stage and may improve the grain yield per plant after drought stress.

TABLE 27
Grain yield analysis of OsLecRK4.1 transgenic rice plants under field drought conditions
	Number of	Number of	Yield per	CK = ZH11-TC	CK= DP0158
Line ID	survival plants	harvested plants	plant (g)	P value	P ≤ 0.1	P value	P≤ 0.1
DP0173.01	40	24	3.47	0.128		0.052	Y
DP0173.04	40	24	4.49	0.792		0.001	Y
DP0173.06	40	24	2.21	0.001		0.867
DP0173.08	39	23	4.69	0.948		0.001	Y
DP0173.11	40	24	3.42	0.045		0.133
DP0173.12	40	24	3.30	0.063		0.105
DP0173.13	39	23	2.15	0.001		0.910
DP0173.14	32	20	2.39	0.001		0.904
DP0173.15	39	21	4.17	0.360		0.010	Y
DP0173.17	40	24	2.91	0.008		0.393
DP0173.18	38	24	3.34	0.045		0.119
DP0173.25	40	24	3.32	0.072		0.076	Y
ZH11-TC	40	24	4.51
DP0158	40	24	2.36
DP0173			3.21	0.031		0.080	Y
(construct)

5) Field DRT Validation Results of OsLecRK4.2 (DP0209) Transgenic Rice

Twelve OsLecRK4.2 transgenic lines were tested in the first field experiment using ZH11-TC and DP0158 rice plants as the controls. Four replicates per transgenic line and ten plants per replicate were planted into the same block. Watering was stopped from panicle initiation stage II to seed mature to produce heavier drought stress. The soil volumetric moisture content decreased from about 35% to 5% during the heading and maturation stages (FIG. 12). Four transgenic lines DP0209.13, DP0209.26, DP0209.28 and DP0209.34 showed greener leaf and less leaf roll degree compared with the ZH11-TC and DP0158 controls planted nearby. DP0209.34 showed better seed setting rate at the maturation stage. The grain weight per plant of OsLecRK4.2 transgenic rice was less than ZH11-TC and DP0158 control at the construct level. Transgenic line level analysis is shown in Table 28. These results demonstrate that OsLecRK4.2 transgenic rice plants had improved drought tolerance at seedling stage after drought stress.

TABLE 28
Grain yield analysis of OsLecRK4.2 transgenic rice plants under field drought conditions
	Number of	Number of	Grain
	survival	harvested	yield per	CK = ZH11-TC	CK = DP0158
Line ID	plants	plants	plant (g)	P value	P ≤ 0.1	P value	P ≤ 0.1
DP0209.02	40	24	2.53	0.062		0.040
DP0209.07	34	22	2.31	0.044		0.030
DP0209.10	40	24	3.28	0.329		0.244
DP0209.11	40	24	3.92	1.000		0.833
DP0209.13	40	25	1.84	0.003		0.001
DP0209.19	40	24	1.86	0.006		0.003
DP0209.25	39	22	3.23	0.349		0.253
DP0209.26	38	24	3.07	0.380		0.287
DP0209.28	32	21	4.85	0.201		0.269
DP0209.30	40	24	2.48	0.061		0.041
DP0209.34	39	24	4.19	0.768		0.933
DP0209.35	40	25	2.49	0.059		0.036
ZH11-TC	40	24	3.66
DP0158	39	24	3.78
DP0209			3.10	0.175		0.115
(construct)

Example 6

Laboratory Paraquat Assays of Transgenic Rice Plants

Paraquat (1,1-dimethyl-4,4-bipyridinium dichloride), is a foliar-applied and non-selective bipyridinium herbicide, and is one of the most widely used herbicides in the world, controlling weeds in a huge variety of crops like corn, rice, soybean etc. In plant cells, paraquat mainly targets to the chloroplasts by accepting electrons from photosystem I and then reacting with oxygen to produce superoxide and hydrogen peroxide, which alters plants' ability to resist photooxidative stress. Drought stress usually leads to increased reactive oxygen species (ROS) in plants and sometimes, the drought tolerance of plant is associated with enhanced antioxidative ability. Paraquat is a potent oxidative stress inducer; it greatly increases the ROS production and inhibits the regeneration of reducing equivalents and compounds necessary for the activity of the antioxidant system. The ROS generation is enhanced under abiotic stress conditions, and the plant responses range from tolerance to death depending on the stress intensity and its associated-ROS levels. Relative low level of paraquat can mimic the stress-associated ROS production and used as a stress tolerance marker in plant stress biology (Hasaneen M. N. A. (2012) Herbicide-Properties, Synthesis and Control of Weeds book). Therefore, the paraquat tolerance of the drought tolerance transgenic rice plants was tested.

Paraquat Assay Methods:

Transgenic rice plants from 8-10 transgenic lines of each transgenic rice line were tested by paraquat assay. Tissue-cultured Zhonghua 11 plants (ZH11-TC) and empty vector transgenic plants (DP0158) were used as controls. T₂ transgenic seeds were sterilized and germinated as described in Example 4, and cultivated in growth room with the temperature of 28-30° C. and humidity of ˜30%. The germinated seeds were placed into a tube with a hole at the bottom, and cultured in water at 30° C. for 5 days till one-leaf and one-terminal bud stage. Uniform seedlings about 3.5-4 cm in height were selected for paraquat testing. Randomized block design was used in this experiment. There were five blocks, each of which has 16×12 holes. Each transgenic line was placed in one row (12 plants/line), and the ZH11-TC and DP0158 controls were placed randomly in 3 rows (3×12 plants) in one block. Then the seedlings were treated with 0.8 μM paraquat solution for 7 days with a 10-h-light/14-h-dark cycle, and the treated seedlings first encountered dark and took up the paraquat solution which was changed every two days. After treated for 7 days, the green seedlings were counted. Those seedlings that maintain green in whole without damage were considered as paraquat tolerant seedlings; while those with bleached leaves or stem were not considered as paraquat tolerant seedling.

Tolerant rate was used as a parameter for this trait screen, which is the percentage of plants which kept green and showed tolerant phenotype over the total plant number.

The data was analyzed at construct level (all transgenic plants compared with the control) and transgenic line level (different transgenic lines compared with the control) using a statistic model of “Y˜seg+line (seg)+rep+error”, random effect of “rep”, and statistic method of “SAS® PROC GLIMMIX”.

Paraquat Assay Results:

1) Paraquat Validation Results of OsGSTU41 (DP0043) Transgenic Rice

For OsGSTU41 transgenic rice, in the first experiment, 231 of the 540 transgenic seedlings (43%) kept green and showed tolerant phenotype after treatment with 0.8 μM paraquat solutions for 7 days, while 48 of the 240 (20%) seedlings from ZH11-TC control and 55 of the 180 (31%) seedlings from DP0158 showed tolerant phenotype under the same condition. The tolerance rate of OsGSTU41 transgenic seedlings was significantly higher than that of ZH11-TC (P value=0.0000) and DP0158 (P value=0.0087) controls. The OsGSTU41 transgenic seedlings grew better after treatment with 0.8 μM paraquat solutions compared with the ZH11-TC and DP0158 seedlings. These results indicate that the OsGSTU41 transgenic seedling exhibited enhanced paraquat tolerance compared with both ZH11-TC and DP0158 controls at construct level.

Further analysis at transgenic line level, as shown in Table 29, indicates that all of the eight OsGSTU41 transgenic lines had greater tolerance rates and five lines showed significantly higher tolerance rates compared with ZH11-TC control; and when compared with the DP0158 control, five lines had greater tolerance rates and four lines showed significantly greater tolerance rates. These results show that over-expression of the OsGSTU41 gene increased the paraquat tolerance or antioxidative ability in the transgenic plants.

TABLE 29
Paraquat tolerance assay of OsGSTU41 transgenic rice plants at transgenic line
level (1st experiment)
	Number of	Number
	tolerant	of total	Tolerancerate	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	(%)	Pvalue	P ≤ 0.05	Pvalue	P ≤ 0.05
DP0043.03	16	60	27	0.2653		0.5700
DP0043.11	16	60	27	0.2653		0.5700
DP0043.12	50	120	42	0.0000	Y	0.0530
DP0043.15	14	60	23	0.5707		0.2900
DP0043.19	30	60	50	0.0000	Y	0.0089	Y
DP0043.25	37	60	62	0.0000	Y	0.0000	Y
DP0043.26	27	60	45	0.0002	Y	0.0465	Y
DP0043.28	41	60	68	0.0000	Y	0.0000	Y
ZH11-TC	48	240	20
DP0158	55	180	31

In the second experiment, 372 of the 600 transgenic seedlings (62%) kept green and showed tolerant phenotype after treated with 0.8 μM paraquat solutions for 7 days, while 86 of the 180 (48%) seedlings from ZH11-TC control and 73 of the 180 (41%) seedlings from DP0158 showed tolerant phenotype under the same condition. The tolerance rate of OsGSTU41 transgenic seedlings was significantly higher than ZH11-TC (P value=0.0008) and DP0158 (P value=0.0000) controls. Analysis at transgenic line level indicates that nine OsGSTU41 transgenic lines had greater tolerance rates than ZH11-TC and DP0158. Five lines showed significantly higher tolerance rates compared with ZH11-TC control, and eight lines showed greater tolerance rates than DP0158 control (Table 30). These results further show that OsGSTU41 transgenic rice had enhanced paraquat tolerance and over-expression of the OsGSTU41 gene increased the paraquat tolerance or antioxidative ability in the transgenic plants.

As described in Example 4 and 5, over-expression of OsGSTU41 gene increased the drought tolerance of rice plants at seedling and mature stage. These cross-validations further confirm that the OsGSTU41 gene plays a role in increasing antioxidative ability and then improve drought tolerance in plant.

TABLE 30
Paraquat tolerance assay of OsGSTU41 transgenic rice plants at transgenic line
level (2nd experiment)
	Number of	Number of
	tolerant	total	Tolerance	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	rate (%)	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0043.03	34	60	57	0.2382		0.0346	Y
DP0043.04	33	60	55	0.3367		0.0567
DP0043.11	36	60	60	0.1072		0.0118	Y
DP0043.12	42	60	70	0.0046	Y	0.0003	Y
DP0043.15	40	60	67	0.0146	Y	0.0010	Y
DP0043.19	41	60	68	0.0083	Y	0.0005	Y
DP0043.21	49	60	82	0.0000	Y	0.0000	Y
DP0043.25	41	60	68	0.0083	Y	0.0005	Y
DP0043.26	21	60	35	0.0909		0.4481
DP0043.28	35	60	58	0.1626		0.0205	Y
ZH11-TC	86	180	48
DP0158	73	180	41

2) Paraquat Validation Results of OsPPCK4 (DP0058) Transgenic Rice

In the first experiment, 380 of the 600 OsPPCK4 transgenic seedlings (63%) kept green and showed tolerant phenotype after 0.8 μM paraquat solution treated for 7 days, while 76 of the 180 (42%) seedlings from ZH11-TC control and 65 of the 180 (36%) seedlings from DP0158 control showed tolerant phenotype. The tolerance rate of all screened OsPPCK4 transgenic seedlings was significantly greater than that of the ZH11-TC (P value=0.0000) and DP0158 (P value=0.0000) controls. The OsPPCK4 transgenic seedlings grew better than ZH11-TC and DP0158 seedlings. All these results show that OsPPCK4 transgenic seedlings exhibited enhanced paraquat tolerance compared with both controls of ZH11-TC and DP0158 seedlings at construct level.

Further analysis at transgenic line level is illustrated in Table 31. Nine lines had greater tolerance rates compared with ZH11-TC and DP0158 controls. The tolerance rates of six lines were significantly greater than that of both ZH11-TC and DP0158 controls. These results demonstrate that OsPPCK4 transgenic rice plants had enhanced paraquat tolerance compared with both controls of ZH11-TC and DP0158 rice plants at construct and transgenic line level at seedling stages.

TABLE 31
Paraquat tolerance assay of OsPPCK4 transgenic rice plants at transgenic line
level (1st experiment)
	Number of	Number of
	tolerant	total	Tolerance	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	rate (%)	Pvalue	P ≤ 0.05	Pvalue	P ≤ 0.05
DP0058.02	51	60	85	0.0000	Y	0.0000	Y
DP0058.03	30	60	50	0.2950		0.0611
DP0058.04	37	60	62	0.0115	Y	0.0011	Y
DP0058.07	34	60	57	0.0561		0.0070	Y
DP0058.10	39	60	65	0.0035	Y	0.0003	Y
DP0058.12	20	60	33	0.2264		0.6964
DP0058.13	53	60	88	0.0000	Y	0.0000	Y
DP0058.14	36	60	60	0.0201	Y	0.0021	Y
DP0058.15	26	60	43	0.8798		0.3195
DP0058.18	54	60	90	0.0000	Y	0.0000	Y
ZH11-TC	76	180	42
DP0158	65	180	36

In the second experiment, 265 of the 540 OsPPCK4 transgenic seedlings (49%) kept green and showed tolerant phenotype after 0.8 μM paraquat solution treated for 7 days, while 88 of the 240 (37%) ZH11-TC seedlings and 75 of the 180 (42%) DP0158 seedlings showed tolerant phenotype at construct level. The paraquat tolerance rate of OsPPCK4 transgenic seedlings was significantly higher than ZH11-TC control (P value=0.0030) and higher than DP0158 control (P value=0.1075) at construct level. These results indicate that the OsPPCK4 transgenic seedlings had enhanced paraquat tolerance at construct level.

The analysis at transgenic line level indicates that five transgenic lines had higher tolerance rates compared with either ZH11-TC or DP0158 controls (Table 32). Five lines had significantly higher tolerance rates than ZH11-TC control, and four lines had significantly higher tolerance rates than DP0158 control. These results demonstrate that OsPPCK4 transgenic rice plants exhibited enhanced paraquat tolerance compared with both ZH11-TC and DP0158 controls at construct and transgenic line level at seedling stage.

TABLE 32
Paraquat tolerance assay of OsPPCK4 transgenic rice plants at transgenic line
level (2nd experiment)
	Number of	Number of
	tolerant	total	Tolerance	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	rate (%)	Pvalue	P ≤ 0.05	Pvalue	P ≤ 0.05
DP0058.02	42	60	70	0.0000	Y	0.0004	Y
DP0058.03	21	60	35	0.8107		0.3628
DP0058.07	23	60	38	0.8104		0.6487
DP0058.10	33	60	55	0.0124	Y	0.0767
DP0058.12	39	60	65	0.0002	Y	0.0029	Y
DP0058.13	36	60	60	0.0019	Y	0.0167	Y
DP0058.14	17	60	28	0.2302		0.0710
DP0058.15	14	60	23	0.0568		0.0142
DP0058.18	40	60	67	0.0001	Y	0.0015	Y
ZH11-TC	88	240	37
DP0158	75	180	42

In the third experiment, 290 of the 540 OsPPCK4 transgenic seedlings (54%) kept green and showed tolerant phenotype after 0.8 μM paraquat solution treated for 7 days, while 77 of the 180 (43%) ZH11-TC seedlings and 89 of the 180 (49%) DP0158 seedlings showed tolerant phenotype at construct level. The paraquat tolerance rate of OsPPCK4 transgenic seedlings was also significantly higher than ZH11-TC control (P value=0.0086) and higher than DP0158 control (P value=0.2236) at construct level.

The analysis at transgenic line level indicates that six transgenic lines had higher tolerance rates compared with either ZH11-TC or DP0158 controls (Table 33). Four lines had significantly higher tolerance rates than ZH11-TC control, and two lines had significantly higher tolerance rates than DP0158 control. These results demonstrate that OsPPCK4 transgenic rice plants exhibited enhanced paraquat tolerance compared with both ZH11-TC and DP0158 controls at construct and transgenic line level at seedling stage. Over-expression of OsPPCK4 gene increased the paraquat tolerance or antioxidative activity of transgenic plants.

TABLE 33
Paraquat tolerance assay of OsPPCK4 transgenic rice plants at transgenic line
level (3rd experiment)
	Number of	Number of	Tolerance
	tolerant	total	rate %	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	(%)	P value	P ≤ 0.05	Pvalue	P ≤ 0.05
DP0058.02	32	60	53	0.1612		0.6038
DP0058.03	37	60	62	0.0146	Y	0.1071
DP0058.07	13	60	22	0.0057		0.0005
DP0058.10	17	60	28	0.0534		0.0067
DP0058.12	35	60	58	0.0418	Y	0.2383
DP0058.13	42	60	70	0.0007	Y	0.0082	Y
DP0058.14	26	60	43	0.9402		0.4154
DP0058.15	34	60	57	0.0678		0.3368
DP0058.18	54	60	90	0.0000	Y	0.0000	Y
ZH11-TC	77	180	43
DP0158	89	180	49

Over-expression of OsPPCK4 gene increased the drought tolerance of transgenic rice plants as illustrated in Example 4 and 5. These cross-validations by three different assays confirm that OsPPCK4 gene increases drought tolerance in plants.

3) Paraquat Validation Results of OsCAM2 (DP0059) Transgenic Rice

In the first experiment, after treated with paraquat solution, 341 of the 480 OsCAM2 transgenic seedlings (71%) kept green and showed tolerant phenotype, whereas only 165 of the 300 ZH11-TC seedlings (55%) and 71 of the 180 DP0158 seedlings (39%) showed paraquat tolerant phenotype. The paraquat tolerance rate of OsCAM2 transgenic plants was significantly higher than that of ZH11-TC (P value=0.0000) and DP0158 (P value=0.0000) controls at construct level. The OsCAM2 transgenic seedlings grew better than either ZH11-TC or DP0158 seedlings after paraquat solution treatment. These results indicate that the OsCAM2 transgenic seedlings had enhanced paraquat tolerance compared with both of ZH11-TC and DP0158 controls at construct level.

The analysis at transgenic line level is displayed in Table 34. All the eight lines had greater tolerance rates than ZH11-TC and DP0158 seedlings, and the eight lines also showed significantly greater paraquat tolerance rates than DP0158. These results further demonstrate over-expression of OsCAM2 gene enhanced the paraquat tolerance in transgenic plants at both construct level and transgenic line level at seedling stage.

TABLE 34
Paraguat tolerance assay of OsCAM2 transgenicrice plants at transgenic line level
(1st experiment)
	Number of	Number
	tolerant	of total	Tolerancerate	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	(%)	Pvalue	P ≤ 0.05	Pvalue	P ≤ 0.05
DP0059.01	35	60	58	0.6370		0.0140	Y
DP0059.04	49	60	82	0.0005	Y	0.0000	Y
DP0059.05	41	60	68	0.0632		0.0003	Y
DP0059.09	42	60	70	0.0376	Y	0.0002	Y
DP0059.11	48	60	80	0.0010	Y	0.0000	Y
DP0059.12	48	60	80	0.0010	Y	0.0000	Y
DP0059.13	38	60	63	0.2404		0.0024	Y
DP0059.14	40	60	67	0.1025		0.0007	Y
ZH11-TC	165	300	55
DP0158	71	180	39

In the second experiment, after treated with paraquat solution, 287 of the 480 OsCAM2 transgenic seedlings (60%) kept green and showed tolerant phenotype, whereas 134 of the 300 ZH11-TC seedlings (45%) and 75 of the 180 DP0158 seedlings (42%) showed paraquat tolerant phenotype. The paraquat tolerance rate of OsCAM2 transgenic plants was significantly higher than that of ZH11-TC (P value=0.0000) and DP0158 (P value=0.0000) controls at construct level. The analysis at transgenic line level is displayed in Table 35. Five lines had greater tolerance rates than ZH11-TC and DP0158 seedlings, and four lines showed significantly greater paraquat tolerance rates than both ZH11-TC and DP0158 controls. These results further demonstrate OsCAM2 transgenic rice exhibited better paraquat tolerance and/or antioxidative activity, and over-expression of OsCAM2 gene enhanced the paraquat tolerance in transgenic plants at both construct level and transgenic line level at seedling stage.

As described in Example 4 and 5, over-expression of OsCAM2 gene can also increase the drought tolerance of the transgenic plants. These cross-validations by two different assays indicate the function of OsCAM2 gene in increasing drought tolerance in plant.

TABLE 35
Paraquat tolerance assay of OsCAM2 transgenicrice plants at transgenic line level
(2nd experiment)
	Number of	Number of
	tolerant	total	Tolerance	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	rate (%)	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0059.01	37	60	62	0.0202	Y	0.0099	Y
DP0059.04	49	60	82	0.0000	Y	0.0000	Y
DP0059.05	27	60	45	0.9618		0.6519
DP0059.09	45	60	75	0.0001	Y	0.0000	Y
DP0059.11	48	60	80	0.0000	Y	0.0000	Y
DP0059.12	31	60	52	0.3249		0.1824
DP0059.13	25	60	42	0.6714		0.9991
DP0059.14	25	60	42	0.6716		0.9989
ZH11-TC	134	300	45
DP0158	75	180	42

4) Paraquat Validation Results of OsDN-DTP4 (DP0167) Transgenic Rice

In the first experiment, construct level analysis of all OsDN-DTP4 transgenic rice plants indicates that, 486 of the 600 seedlings (81%) kept green and showed tolerant phenotype, while 101 of the 180 (56%) ZH11-TC seedlings showed tolerant phenotype, and 71 of 180 (39%) DP0158 seedlings showed tolerant phenotype. The tolerance rate of all tested OsDN-DTP4 transgenic seedlings was significantly higher than that of the ZH11-TC (P value=0.0000) and DP0158 (P value=0.0000) controls. These results indicate that the OsDN-DTP4 transgenic seedlings grew better and enhanced paraquat tolerance compared with either ZH11-TC or DP0158 seedlings at construct level after treated by 0.8 μM paraquat solutions.

Further analysis at transgenic line level indicates that all of the ten lines had greater tolerance rates compared with ZH11-TC and DP0158 controls (Table 36). Nine lines compared with ZH11-TC control and ten lines compared with DP0158 control had significantly higher tolerance rates, respectively. These results demonstrate that OsDN-DTP4 transgenic rice plants exhibited enhanced paraquat tolerance compared with both ZH11-TC and DP0158 controls at construct and transgenic line level at seedling stage.

TABLE 36
Paraquat tolerance assay of OsDN-DTP4 transgenic rice plant at transgenic line
level (1st experiment)
	Number of	Number	Tolerance
	tolerant	of total	rate	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	(%)	Pvalue	P ≤ 0.05	Pvalue	P ≤ 0.05
DP0167.01	52	60	87	0.0002	Y	0.0000	Y
DP0167.02	52	60	87	0.0002	Y	0.0000	Y
DP0167.03	48	60	80	0.0019	Y	0.0000	Y
DP0167.04	48	60	80	0.0019	Y	0.0000	Y
DP0167.05	52	60	87	0.0002	Y	0.0000	Y
DP0167.06	47	60	78	0.0037	Y	0.0000	Y
DP0167.07	46	60	77	0.0068	Y	0.0000	Y
DP0167.11	40	60	67	0.1532		0.0000	Y
DP0167.12	46	60	77	0.0068	Y	0.0000	Y
DP0167.13	55	60	92	0.0000	Y	0.0000	Y
ZH11-TC	101	180	56
DP0158	71	180	39

In the second experiment, 366 of the 600 seedlings (61%) kept green and showed tolerant phenotype, while 63 of the 180 (35%) ZH11-TC seedlings showed tolerant phenotype, and 98 of the 180 (54%) DP0158 seedlings showed tolerant phenotype. The tolerance rate of all tested OsDN-DTP4 transgenic seedlings was significantly higher than ZH11-TC (P value=0.0000) and DP0158 (P value=0.0491) controls. These results indicate that the OsDN-DTP4 transgenic seedlings grew better and enhanced paraquat tolerance compared with either ZH11-TC or DP0158 seedlings at construct level.

Analysis at transgenic line level indicates that eight lines exhibited greater tolerance rates compared with ZH11-TC and DP0158 controls (Table 37). Eight lines compared with ZH11-TC control and two lines compared with DP0158 control had significantly higher tolerance rates, respectively. These results further demonstrate that OsDN-DTP4 transgenic rice plants exhibited enhanced paraquat tolerance and/or antioxidative activity at seedling stage. Over-expression of OsDN-DTP4 gene increased the paraquat tolerance and/or antioxidative activity in transgenic plants.

As described in Example 4, over-expression of OsDN-DTP4 can also increase the drought tolerance of the transgenic plants. These cross-validations by two different assays demonstrate that OsDN-DTP4 expression increased drought tolerance in plant.

TABLE 37
Paraquat tolerance assay of OsDN-DTP4 transgenic rice plant at transgenic line
level (2nd experiment)
	Number of	Number of
	tolerant	total	Tolerance	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	rate (%)	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0167.02	39	60	65	0.0002	Y	0.1573
DP0167.03	37	60	62	0.0007	Y	0.3313
DP0167.04	44	60	73	0.0000	Y	0.0132	Y
DP0167.05	36	60	60	0.0013	Y	0.4542
DP0167.06	24	60	40	0.4863		0.0578
DP0167.07	36	60	60	0.0013	Y	0.4542
DP0167.11	38	60	63	0.0004	Y	0.2326
DP0167.12	41	60	68	0.0000	Y	0.0645
DP0167.13	43	60	72	0.0000	Y	0.0234	Y
ZH11-TC	63	180	35
DP0158	98	180	54

5) Paraquat Validation Results of OsLecRK4.1 (DP0173) Transgenic Rice

For OsLecRK4.1 transgenic rice, in the first experiment, 462 of the 600 OsLecRK4.1 transgenic seedlings (77%) kept green and showed tolerant phenotype after treated with paraquat solutions, whereas 99 of the 180 (55%) ZH11-TC seedlings and 95 of the 180 (53%) DP0158 seedlings showed paraquat tolerant phenotype respectively. The tolerance rate of OsLecRK4.1 transgenic seedlings was significantly higher than that of ZH11-TC (P value=0.0000) and DP0158 (P value=0.0000) controls. The OsLecRK4.1 transgenic seedlings grew better after paraquat solution treatment compared with the ZH11-TC or DP0158 seedlings. All these results demonstrate that OsLecRK4.1 transgenic seedlings exhibited enhanced paraquat tolerance compared with both of ZH11-TC and DP0158 controls at construct level.

As shown in Table 38, nine of the ten OsLecRK4.1 transgenic lines had higher tolerance rates than ZH11-TC and DP0158 controls at transgenic line level. And seven lines compared with ZH11-TC control and 8 lines compared with DP0158 control had significantly higher tolerance rates. These results further demonstrate that over-expression of OsLecRK4.1 gene increased paraquat tolerance or antioxidative activity of transgenic rice plants.

TABLE 38
Paraquat tolerance assay of OsLecRK4.1 transgenic rice plant at transgenic line
level (1st experiment)
	Number of	Number of
	tolerant	total	Tolerancerate	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	(%)	Pvalue	P ≤ 0.05	Pvalue	P ≤ 0.05
DP0173.01	48	60	80	0.0014	Y	0.0007	Y
DP0173.02	34	60	57	0.8227		0.6027
DP0173.04	41	60	68	0.0762		0.0408	Y
DP0173.05	30	60	50	0.5039		0.7105
DP0173.06	50	60	83	0.0004	Y	0.0002	Y
DP0173.11	50	60	83	0.0004	Y	0.0002	Y
DP0173.12	58	60	97	0.0000	Y	0.0000	Y
DP0173.13	45	60	75	0.0091	Y	0.0043	Y
DP0173.14	54	60	90	0.0000	Y	0.0000	Y
DP0173.15	52	60	87	0.0001	Y	0.0000	Y
ZH11-TC	99	180	55
DP0158	95	180	53

In the second experiment, 425 of the 600 OsLecRK4.1 transgenic seedlings (71%) kept green and showed tolerant phenotype after treated with paraquat solutions, while 103 of the 180 (57%) ZH11-TC seedlings and 108 of the 180 (60%) DP0158 seedlings showed paraquat tolerant phenotype respectively. The tolerance rate of OsLecRK4.1 transgenic seedlings was significantly higher than ZH11-TC (P value=0.0002) and DP0158 (P value=0.0020) controls. Analysis at transgenic line level shows that nine transgenic lines had higher tolerance rates than ZH11-TC and DP0158 controls, four lines compared with ZH11-TC control and three lines compared with DP0158 control exhibited significantly higher tolerance rates (Table 39). These results further demonstrate that over-expression of OsLecRK4.1 gene increased paraquat tolerance or antioxidative activity of transgenic rice plants.

Over-expression of OsLecRK4.1 gene enhanced the drought tolerance of the transgenic rice plants as described in Example 5. These cross-validations by two different assays indicate that OsLecRK4.1 gene functions in increasing drought tolerance in plant.

TABLE 39
Paraquat tolerance assay of OsLecRK4.1 transgenic rice plant at transgenic line
level (2nd experiment)
	Number of	Number of
	tolerant	total	Tolerance	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	rate (%)	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0173.01	55	60	92	0.0000	Y	0.0001	Y
DP0173.02	43	60	72	0.0523		0.1105
DP0173.04	44	60	73	0.0311	Y	0.0689
DP0173.05	37	60	62	0.5460		0.8196
DP0173.06	38	60	63	0.4066		0.6475
DP0173.11	36	60	60	0.7060		0.9996
DP0173.12	47	60	78	0.0055	Y	0.0136	Y
DP0173.13	31	60	52	0.4543		0.2606
DP0173.14	53	60	88	0.0001	Y	0.0003	Y
DP0173.15	41	60	68	0.1325		0.2519
ZH11-TC	103	180	57
DP0158	108	180	60

6) Paraquat Validation Results of OsLecRK4.2 (DP0209) Transgenic Rice

In the first experiment, 600 OsLecRK4.2 transgenic seedlings were considered as a whole and analyzed at construct level. 221 of the 600 OsLecRK4.2 transgenic seedlings (37%) kept green and showed tolerant phenotype, while only 30 of the 240 (17%) ZH11-TC seedlings and 29 of the 180 (16%) DP0158 seedlings showed tolerant phenotype. The paraquat tolerance rate of OsLecRK4.2 transgenic seedlings was significantly higher than that of ZH11-TC (P value=0.0000) and DP0158 controls (P value=0.0000) at construct level. These results indicate that OsLecRK4.2 transgenic seedlings had enhanced paraquat tolerance at construct level, and the OsLecRK4.2 transgenic seedlings grew better after treated by 0.8 μM paraquat solutions compared with the ZH11-TC and DP0158 seedlings.

The analysis at transgenic line level indicates that nine of the ten tested transgenic lines had higher tolerance rates compared with either ZH11-TC or DP0158 controls (Table 40). Eight lines showed significantly higher tolerance rates than that of ZH11-TC and DP0158 controls respectively as shown in Table 37. These results demonstrate that OsLecRK4.2 transgenic rice plants exhibited enhanced paraquat tolerance compared with both ZH11-TC and DP0158 controls at construct and transgenic line level at seedling stage. Over-expression of OsLecRK4.2 gene increased the paraquat tolerance or antioxidative activity in transgenic plants.

TABLE 40
Paraquat tolerance assay of OsLecRK4.2 transgenic rice plant at transgenic line
level (1st experiment)
	Number of	Number of
	tolerant	total	Tolerancerate	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	rate (%)	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0209.01	32	60	53	0.0000	Y	0.0000	Y
DP0209.02	22	60	37	0.0022	Y	0.0016	Y
DP0209.05	22	60	37	0.0022	Y	0.0016	Y
DP0209.06	23	60	38	0.0011	Y	0.0008	Y
DP0209.08	27	60	45	0.0000	Y	0.0000	Y
DP0209.09	31	60	52	0.0000	Y	0.0000	Y
DP0209.10	7	60	12	0.3576		0.4075
DP0209.11	22	60	37	0.0022		0.0016	Y
DP0209.12	14	60	23	0.2526	Y	0.2120
DP0209.14	21	60	35	0.0044	Y	0.0032	Y
ZH11-TC	30	180	17
DP0158	29	180	16

In the second experiment, 351 of the 600 OsLecRK4.2 transgenic seedlings (59%) kept green and showed tolerant phenotype, while 66 of the 180 (37%) ZH11-TC seedlings and 85 of the 180 (47%) DP0158 seedlings showed tolerant phenotype. The paraquat tolerance rate of OsLecRK4.2 transgenic seedlings was significantly higher than ZH11-TC (P value=0.0000) and DP0158 controls (P value=0.0080) at construct level. These results indicate that OsLecRK4.2 transgenic seedlings had enhanced paraquat tolerance at construct level.

The analysis at transgenic line level indicates that eight lines exhibited higher tolerance rates compared with either ZH11-TC or DP0158 controls (Table 41). Eight lines showed significantly higher tolerance rates than ZH11-TC and five lines exhibited significantly higher tolerance rates than DP0158 controls respectively. These results further demonstrate that OsLecRK4.2 transgenic rice plants exhibited enhanced paraquat tolerance at seedling stage. Over-expression of OsLecRK4.2 gene increased the paraquat tolerance or antioxidative activity in transgenic plants.

Over-expression of OsLecRK4.2 gene can also increase the drought tolerance of transgenic rice plants as illustrated in Example 5. These cross-validations by two different assays confirm that OsLecRK4.2 gene can increase drought tolerance in plants.

TABLE 41
Paraquat tolerance assay of OsLecRK4.2 transgenic rice plant at transgenic line
level (2nd experiment)
	Number of	Number of
	tolerant	total	Tolerance	CK = ZH11-TC	CK = DP0158
Line ID	seedlings	seedlings	rate (%)	P value	P ≤ 0.05	P value	P ≤ 0.05
DP0209.01	42	60	70	0.0000	Y	0.0038	Y
DP0209.02	22	60	37	1.0000		0.1607
DP0209.05	35	60	58	0.0051	Y	0.1424
DP0209.06	33	60	55	0.0160	Y	0.3014
DP0209.08	31	60	52	0.0458	Y	0.5531
DP0209.09	44	60	73	0.0000	Y	0.0011	Y
DP0209.10	24	60	40	0.6458		0.3350
DP0209.11	43	60	72	0.0000	Y	0.0020	Y
DP0209.12	38	60	63	0.0008	Y	0.0358	Y
DP0209.14	39	60	65	0.0004	Y	0.0212	Y
ZH11-TC	66	180	37
DP0158	85	180	47

Example 7

Transformation and Evaluation of Maize with Rice Drought Tolerance Genes

Maize plants can be transformed to over-express Oryza sativa drought tolerance genes or a corresponding homolog from maize, Arabidopsis, or other species. Expression of the gene in the maize transformation vector can be under control of a constitutive promoter such as the maize ubiquitin promoter (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689) or under control of another promoter, such as a stress-responsive promoter or a tissue-preferred promoter. The recombinant DNA construct can be introduced into maize cells by particle bombardment substantially as described in International Patent Publication WO 2009/006276. Alternatively, maize plants can be transformed with the recombinant DNA construct by Agrobacterium-mediated transformation substantially as described by Zhao et al. in Meth. Mol. Biol. 318:315-323 (2006) and in Zhao et al., Mol. Breed. 8:323-333 (2001) and U.S. Pat. No. 5,981,840 issued Nov. 9, 1999. The Agrobacterium-mediated transformation process involves bacterium inoculation, co-cultivation, resting, selection and plant regeneration.

Progeny of the regenerated plants, such as T₁ plants, can be subjected to a soil-based drought stress. Using image analysis, plant area, volume, growth rate and color can be measured at multiple times before and during drought stress. Significant delay in wilting or leaf area reduction, a reduced yellow-color accumulation, and/or an increased growth rate during drought stress, relative to a control, will be considered evidence that the gene functions in maize to enhance drought tolerance.

Example 8

Transformation and Evaluation of Gaspe Flint Derived Maize Lines

As described in Example 7, maize plants can be transformed to over-express the rice drought tolerance genes, or corresponding homologs from another species. In certain circumstances, recipient plant cells can be from a uniform maize line having a short life cycle (“fast cycling”), a reduced size, and high transformation potential, and are disclosed in Tomes et al. U.S. Pat. No. 7,928,287.

The population of transgenic (T₀) plants resulting from the transformed maize embryos can be grown in a controlled greenhouse environment using a modified randomized block design to reduce or eliminate environmental error. For example, a group of 30 plants, comprising 24 transformed experimental plants and 6 control plants (collectively, a “replicate group”), are placed in pots which are arranged in an array (a.k.a. a replicate group or block) on a table located inside a greenhouse. Each plant, control or experimental, is randomly assigned to a location with the block which is mapped to a unique, physical greenhouse location as well as to the replicate group. Multiple replicate groups of 30 plants each may be grown in the same greenhouse in a single experiment. The layout (arrangement) of the replicate groups should be determined to minimize space requirements as well as environmental effects within the greenhouse. Such a layout may be referred to as a compressed greenhouse layout.

Each plant in the line population is identified and tracked throughout the evaluation process, and the data gathered from that plant are automatically associated with that plant so that the gathered data can be associated with the transgene carried by the plant. For example, each plant container can have a machine readable label (such as a Universal Product Code (UPC) bar code) which includes information about the plant identity, which in turn is correlated to a greenhouse location so that data obtained from the plant can be automatically associated with that plant.

Alternatively any efficient, machine readable, plant identification system can be used, such as two-dimensional matrix codes or even radio frequency identification tags (RFID) in which the data is received and interpreted by a radio frequency receiver/processor (U.S. Pat. Nos. 7,403,855 and 7,702,462).

Each greenhouse plant in the T₀ line population, including any control plants, is analyzed for agronomic characteristics of interest, and the agronomic data for each plant are recorded or stored in a manner so as to be associated with the identifying data for that plant. Confirmation of a phenotype (gene effect) can be accomplished in the T₁ generation with a similar experimental design to that described above.

Example 9

Laboratory Drought Assays of Rice Drought Tolerance Genes in Arabidopsis

To understand whether rice drought tolerance genes can improve dicot plants' drought tolerance, or other traits, the rice drought tolerance gene over-expression vectors were transformed into Arabidopsis (Columbia) using floral dip method by Agrobacterium mediated transformation procedure and transgenic plants were identified (Clough, S. T. and Bent, A. F. (1998) The Plant Journal 16, 735-743; Zhang, X. et al. (2006) Nature Protocols 1: 641-646).

A 16.8-kb T-DNA based binary vector which is called pBC-yellow was used in this experiment. This vector contains the RD29a promoter driving expression of the gene for ZS-Yellow, which confers yellow fluorescence to transformed seed. The rice tolerance genes were cloned as described in Example 1, and constructed in the Gateway vector. Then using the INVITROGEN™ GATEWAY® technology, an LR Recombination Reaction was performed on the entry clone containing the directionally cloned PCR product and the pBC-yellow vector, and the over-expression vectors were obtained.

T₂ seeds were used for lab drought assay. Arabidopsis drought screening is a soil-based water withdrawal assay performed in a growth chamber with conditions of light intensity 145 μMol, temperature 22° C. day/20° C. night and humidity ˜60%. The transgenic seeds were sorted by COPAS™ (Complex Object Parametric Analyzer and Sorter, a seed sorter, Union Biometrica), and were stratified by putting in 0.1% agarose solution, and placing at 4° C. for 3 days. Wild-type Arabidopsis were used as control and stratified as above. 36 plants each for over-expression transgenic Arabidopsis and wild-type were planted equidistantly and alternatively to each other in a zig-zag fashion. The soil composition was 3 parts peat moss, 2 parts vermiculite and 1 part perlite. Apart from these, fertilizers and fungicides were added to the soil in the following concentrations: NPK (Nitrogen, Phosphorus, Potassium)—1 gm/kg soil, Micronutrients—0.5 gm/kg soil, Fungicide—0.5 gm/kg soil. Plants were thinned to 9 plants per pot (72 plants per flat), and were well watered for the first 12 days, then saturated with 1 L of deionized water for 30 min with excess water drained off completely. The plants were imaged between days 28 and 36 after germination using an imaging device and data were analyzed. The flats were rotated each day from the second day after sowing till the last day of imaging. The files generated in the imaging device were converted into XLS files and put in a Stan's format and sent to ESL for generating Stan's score for the experimental lines. Rate of decay or wilting under drought conditions is used as tested parameter. The cut-off Score=1.5.

Claims

1. A method of increasing drought tolerance in a plant, the method comprising: (a) introducing into a plant cell, plant, or plant part a construct comprising a polynucleotide operably linked to at least one heterologous regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 5; and (b) selecting a plant cell, plant, or plant part comprising the construct for increased drought tolerance as compared to a control plant lacking the construct.

2. The method of claim 1, wherein said plant is selected from the group consisting of rice, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, barley, millet, sugar cane and switchgrass.

3. The method of claim 1, wherein the plant is maize or rice.

4. The plant method of claim 1, wherein the polynucleotide is expressed in the root tissue.

5. The method of claim 1, wherein the selection is for increased yield.

6. The method of claim 1, wherein the selection occurs during the grain fill stage.